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Monoclonal Antibody Western Blotting Sister Chromatid Segregation

Also showing Monoclonal Antibody Western Blotting Mitotic Sister Chromatid Segregation

$303
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse

Application Methods: Western Blotting

Background: Structural maintenance of chromosomes 1 (SMC1) protein is a chromosomal protein member of the cohesin complex that enables sister chromatid cohesion and plays a role in DNA repair (1,2). ATM/NBS1-dependent phosphorylation of SMC1 occurs at Ser957 and Ser966 in response to ionizing radiation (IR) as part of the intra-S-phase DNA damage checkpoint (3). SMC1 phosphorylation is ATM-independent in cells subjected to other forms of DNA damage, including UV light and hydroxyurea treatment (4). While phosphorylation of SMC1 is required for activation of the IR-induced intra-S-phase checkpoint, the precise mechanism is not well understood and may involve a conformational change that affects SMC1-SMC3 interaction (3).

$111
20 µl
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: The cohesin complex consists of a heterodimer between SMC1 (SMC1A or B) and SMC3, bound by additional RAD21 and STAG proteins (STAG1, 2, or 3) (1,2). These proteins form a ring-like structure that mediates the cohesion of two sister chromatids after DNA replication in S phase (1,2). RAD21 and STAG2 are phosphorylated by Polo-like kinase (PLK) during prophase, which leads to the dissociation of cohesin complexes from the chromosome arms; however, cohesin remains bound to centromeres until anaphase (3,4). RAD21 is cleaved by separin/ESPL1 in anaphase, which leads to dissociation of the remaining cohesin from centromeres, enabling sister chromatids to segregate during mitosis (5). RAD21 is also cleaved by caspase-3 and caspase-7 during apoptosis, resulting in a 64 kDa carboxy-terminal cleavage product that translocates to the cytoplasm and may help to trigger apoptosis (6,7). In addition to mediating cohesion of sister chromatids, the cohesin complex plays important roles in gene regulation and DNA repair, as SMC1 and SMC3 are both phosphorylated by ATM and ATR kinases upon DNA damage (1,2).

$303
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: Structural maintenance of chromosomes 1 (SMC1) protein is a chromosomal protein member of the cohesin complex that enables sister chromatid cohesion and plays a role in DNA repair (1,2). ATM/NBS1-dependent phosphorylation of SMC1 occurs at Ser957 and Ser966 in response to ionizing radiation (IR) as part of the intra-S-phase DNA damage checkpoint (3). SMC1 phosphorylation is ATM-independent in cells subjected to other forms of DNA damage, including UV light and hydroxyurea treatment (4). While phosphorylation of SMC1 is required for activation of the IR-induced intra-S-phase checkpoint, the precise mechanism is not well understood and may involve a conformational change that affects SMC1-SMC3 interaction (3).

$111
20 µl
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Rat

Application Methods: Immunohistochemistry (Paraffin), Immunoprecipitation, Western Blotting

Background: At least four distinct polo-like kinases exist in mammalian cells: PLK1, PLK2, PLK3, and PLK4/SAK (1). PLK1 apparently plays many roles during mitosis, particularly in regulating mitotic entry and exit. The mitosis promoting factor (MPF), cdc2/cyclin B1, is activated by dephosphorylation of cdc2 (Thr14/Tyr15) by cdc25C. PLK1 phosphorylates cdc25C at Ser198 and cyclin B1 at Ser133 causing translocation of these proteins from the cytoplasm to the nucleus (2-5). PLK1 phosphorylation of Myt1 at Ser426 and Thr495 has been proposed to inactivate Myt1, one of the kinases known to phosphorylate cdc2 at Thr14/Tyr15 (6). Polo-like kinases also phosphorylate the cohesin subunit SCC1, causing cohesin displacement from chromosome arms that allow for proper cohesin localization to centromeres (7). Mitotic exit requires activation of the anaphase promoting complex (APC) (8), a ubiquitin ligase responsible for removal of cohesin at centromeres, and degradation of securin, cyclin A, cyclin B1, Aurora A, and cdc20 (9). PLK1 phosphorylation of the APC subunits Apc1, cdc16, and cdc27 has been demonstrated in vitro and has been proposed as a mechanism by which mitotic exit is regulated (10,11).Substitution of Thr210 with Asp has been reported to elevate PLK1 kinase activity and delay/arrest cells in mitosis, while a Ser137Asp substitution leads to S-phase arrest (12). In addition, while DNA damage has been found to inhibit PLK1 kinase activity, the Thr210Asp mutant is resistant to this inhibition (13). PLK1 has been reported to be phosphorylated in vivo at Ser137 and Thr210 in mitosis; DNA damage prevents phosphorylation at these sites (14).

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunoprecipitation, Western Blotting

Background: At least four distinct polo-like kinases exist in mammalian cells: PLK1, PLK2, PLK3, and PLK4/SAK (1). PLK1 apparently plays many roles during mitosis, particularly in regulating mitotic entry and exit. The mitosis promoting factor (MPF), cdc2/cyclin B1, is activated by dephosphorylation of cdc2 (Thr14/Tyr15) by cdc25C. PLK1 phosphorylates cdc25C at Ser198 and cyclin B1 at Ser133 causing translocation of these proteins from the cytoplasm to the nucleus (2-5). PLK1 phosphorylation of Myt1 at Ser426 and Thr495 has been proposed to inactivate Myt1, one of the kinases known to phosphorylate cdc2 at Thr14/Tyr15 (6). Polo-like kinases also phosphorylate the cohesin subunit SCC1, causing cohesin displacement from chromosome arms that allow for proper cohesin localization to centromeres (7). Mitotic exit requires activation of the anaphase promoting complex (APC) (8), a ubiquitin ligase responsible for removal of cohesin at centromeres, and degradation of securin, cyclin A, cyclin B1, Aurora A, and cdc20 (9). PLK1 phosphorylation of the APC subunits Apc1, cdc16, and cdc27 has been demonstrated in vitro and has been proposed as a mechanism by which mitotic exit is regulated (10,11).Substitution of Thr210 with Asp has been reported to elevate PLK1 kinase activity and delay/arrest cells in mitosis, while a Ser137Asp substitution leads to S-phase arrest (12). In addition, while DNA damage has been found to inhibit PLK1 kinase activity, the Thr210Asp mutant is resistant to this inhibition (13). PLK1 has been reported to be phosphorylated in vivo at Ser137 and Thr210 in mitosis; DNA damage prevents phosphorylation at these sites (14).

$129
20 µl
$303
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunohistochemistry (Paraffin), Western Blotting

Background: DNA topoisomerases I and II are nuclear enzymes; type II consists of two highly homologous isoforms: topoisomerase IIα and IIβ. These enzymes regulate the topology of DNA, maintain genomic integrity, and are essential for processes such as DNA replication, recombination, transcription, and chromosome segregation by allowing DNA strands to pass through each other (1). Topoisomerase I nicks and rejoins one strand of the duplex DNA, while topoisomerase II transiently breaks and closes double-stranded DNA (2). Topoisomerases are very susceptible to various stresses. Acidic pH or oxidative stress can convert topoisomerases to DNA-breaking nucleases, causing genomic instability and cell death. DNA-damaging topoisomerase targeting drugs (e.g., etoposide) also convert topoisomerases to nucleases, with the enzyme usually trapped as an intermediate that is covalently bound to the 5+ end of the cleaved DNA strand(s). Research studies have shown that this intermediate leads to genomic instability and cell death. Thus, agents that target topoisomerases are highly sought after cancer chemotherapeutic drugs (3). Ca2+-regulated phosphorylation of topoisomerase IIα at Ser1106 modulates the activity of this enzyme and its sensitivity to targeting drugs (4).

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: DNA topoisomerases I and II are nuclear enzymes; type II consists of two highly homologous isoforms: topoisomerase IIα and IIβ. These enzymes regulate the topology of DNA, maintain genomic integrity, and are essential for processes such as DNA replication, recombination, transcription, and chromosome segregation by allowing DNA strands to pass through each other (1). Topoisomerase I nicks and rejoins one strand of the duplex DNA, while topoisomerase II transiently breaks and closes double-stranded DNA (2). Topoisomerases are very susceptible to various stresses. Acidic pH or oxidative stress can convert topoisomerases to DNA-breaking nucleases, causing genomic instability and cell death. DNA-damaging topoisomerase targeting drugs (e.g., etoposide) also convert topoisomerases to nucleases, with the enzyme usually trapped as an intermediate that is covalently bound to the 5+ end of the cleaved DNA strand(s). Research studies have shown that this intermediate leads to genomic instability and cell death. Thus, agents that target topoisomerases are highly sought after cancer chemotherapeutic drugs (3). Ca2+-regulated phosphorylation of topoisomerase IIα at Ser1106 modulates the activity of this enzyme and its sensitivity to targeting drugs (4).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: Structural maintenance of chromosomes 2 (SMC2) and 4 (SMC4) proteins are subunits of the condensin complex, which enables chromosome condensation and maintains the compaction of chromosomes as they separate to opposite poles during anaphase (1-3). In addition to regulating chromosome condensation, condensin is a general regulator of chromosome architecture and may function to regulate gene expression and DNA repair. SMC proteins contain a hallmark bipartite ATPase domain of the ABC ATPase superfamily, which consists of an N-terminal Walker A motif nucleotide-binding domain and C-terminal Walker B motif catalytic domain that interact to form a functional ATPase (1-3). The two ATPase domains are connected by two coiled coil domains separated by a central hinge region that facilitates protein-protein interactions between partnering SMC proteins. In the case of the condensin complex, SMC2 and SMC4 interact to form a functional ATPase required for chromatin condensation; however, the mechanism by which this ATPase activity regulates chromsome architecture is still being determined. In addition to SMC proteins, condensin contains three auxiliary subunits, which function to regulate condensin ATPase activity. Higher eukaryotes contain two distinct condensin complexes (condensin I and II), both of which contain SMC2 and SMC4 (1-3). Condensin I also contains the auxiliary subunits CAP-D2, CAP-G and CAP-H, while condensin II contains the related auxiliary proteins CAP-D3, CAP-G2 and CAP-H2. The two condensin complexes show different localization patterns during the cell cycle and on chromosomes and both are required for successful mitosis, suggesting distinct functions for each complex (1-3).

$303
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse

Application Methods: Western Blotting

Background: The Large tumor suppressor (LATS) proteins (LATS1, LATS2) are serine/threonine kinases that belong to the NDR family (1). The Drosophila homolog (warts) was first identified as a tumor suppressor protein that plays a role in the maintenance of ploidy. Human LATS1 was shown to localize to the centrosome and the mitotic spindle and control G2/M transition by negatively regulating cdc2 kinase activity (2,3). LATS1 is also reported to play a role in the G1 tetraploidy checkpoint, via control of p53 expression (4). LATS1 affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1 (5). LATS1 also binds the phosphorylated form of zyxin, a regulator of actin filament assembly. This interaction promotes localization of zyxin to the mitotic spindle, suggesting a role for actin regulatory proteins during mitosis (6). Decreased expression of LATS1 is associated with breast tumor aggressiveness (7), and mutations perturbing LATS1 have been associated with human sarcomas and ovarian sarcomas (8,9). LATS1 knockout mice develop soft-tissue sarcomas, ovarian stromal cell tumor, and display a high sensitivity to carcinogenic treatments (10). LATS1 and LATS2 have also been identified as key members of the Hippo signaling pathway, a conserved kinase cascade that functions to regulate cell growth and apoptosis (11). Phosphorylation of LATS by Mammalian Sterile-20-like proteins (e.g., MST1) results in LATS-mediated phosphorylation of the transcriptional co-activators YAP and TAZ (12, 13). LATS-mediated phosphorylation of YAP and TAZ promotes their cytoplasmic sequestration and association with 14-3-3 proteins, and subsequent proteasomal degradation, leading to downregulation of YAP/TAZ target genes that promote cell growth (11, 14).

$111
20 µl
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse

Application Methods: Immunoprecipitation, Western Blotting

Background: The Large tumor suppressor (LATS) proteins (LATS1, LATS2) are serine/threonine kinases that belong to the NDR family (1). The Drosophila homolog (warts) was first identified as a tumor suppressor protein that plays a role in the maintenance of ploidy. Human LATS1 was shown to localize to the centrosome and the mitotic spindle and control G2/M transition by negatively regulating cdc2 kinase activity (2,3). LATS1 is also reported to play a role in the G1 tetraploidy checkpoint, via control of p53 expression (4). LATS1 affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1 (5). LATS1 also binds the phosphorylated form of zyxin, a regulator of actin filament assembly. This interaction promotes localization of zyxin to the mitotic spindle, suggesting a role for actin regulatory proteins during mitosis (6). Decreased expression of LATS1 is associated with breast tumor aggressiveness (7), and mutations perturbing LATS1 have been associated with human sarcomas and ovarian sarcomas (8,9). LATS1 knockout mice develop soft-tissue sarcomas, ovarian stromal cell tumor, and display a high sensitivity to carcinogenic treatments (10). LATS1 and LATS2 have also been identified as key members of the Hippo signaling pathway, a conserved kinase cascade that functions to regulate cell growth and apoptosis (11). Phosphorylation of LATS by Mammalian Sterile-20-like proteins (e.g., MST1) results in LATS-mediated phosphorylation of the transcriptional co-activators YAP and TAZ (12, 13). LATS-mediated phosphorylation of YAP and TAZ promotes their cytoplasmic sequestration and association with 14-3-3 proteins, and subsequent proteasomal degradation, leading to downregulation of YAP/TAZ target genes that promote cell growth (11, 14).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: The Mitotic Checkpoint Complex (MCC), which contains Bub1, Bub1b, Bub3, Mad2, and Cdc20, controls chromosome segregation and monitors kinetochore-microtubule interactions (1). During mitosis, the MCC complex inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex/Cyclosome (APC/C), thereby preventing cells with unaligned chromosomes from prematurely entering anaphase (2). Research studies have shown that Bub1b and Bub1 kinases are mutated in several types of human malignancies including hematopoietic, colorectal, lung, and breast cancers (3). Biallelic mutations in Bub1b have been found in mosaic variegated aneuploidy syndrome and premature chromatid separation syndrome (4). Bub1b mouse germline knockouts are embryonic lethal with heterozygous animals displaying genetic instability, early aging phenotypes, and increased cancer susceptibility (5). Bub3 binds both Bub1 and Bub1b, facilitating their recruitment to kinetochores (6), and is required for functional microtubule-kinetochore interactions (7).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: Correct segregation of sister chromatids prior to the onset of cell division is essential to the maintenance of genetic integrity and the avoidance of aneuploidy and chromosomal instability, characteristics of many cancer cells. The mitotic checkpoint, also known as the spindle assembly checkpoint, monitors accurate attachment of kinetochores to the spindle, inhibits mitosis and delays the onset of anaphase until all chromosomes are aligned at the metaphase plate (1). MAD2L1 is an essential participant in the mitotic checkpoint (2). It exists in two conformations, including the open and inactive O-MAD2 form and the closed, active C-MAD2 form. Prior to mitosis, MAD2L1 is localized to the cytosol and exists largely in the closed, inactive form. During the mitotic checkpoint, MAD2L1 switches to the open, active conformation (3). Together with other checkpoint proteins, MAD2L1 binds to and deactivates Cdc20, thereby inhibiting the anaphase promoting complex (4). When the kinetochores are correctly attached to the spindle, MAD2L1 releases Cdc20, which allows activation of the anaphase promoting complex and subsequent degradation of key mitotic substrates and the initiation of metaphase-anaphase transition (5).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: The Mitotic Checkpoint Complex (MCC), which contains Bub1, Bub1b, Bub3, Mad2, and Cdc20, controls chromosome segregation and monitors kinetochore-microtubule interactions (1). During mitosis, the MCC complex inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex/Cyclosome (APC/C), thereby preventing cells with unaligned chromosomes from prematurely entering anaphase (2). Research studies have shown that Bub1b and Bub1 kinases are mutated in several types of human malignancies including hematopoietic, colorectal, lung, and breast cancers (3). Biallelic mutations in Bub1b have been found in mosaic variegated aneuploidy syndrome and premature chromatid separation syndrome (4). Bub1b mouse germline knockouts are embryonic lethal with heterozygous animals displaying genetic instability, early aging phenotypes, and increased cancer susceptibility (5). Bub3 binds both Bub1 and Bub1b, facilitating their recruitment to kinetochores (6), and is required for functional microtubule-kinetochore interactions (7).

$111
20 µl
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Immunohistochemistry (Paraffin), Western Blotting

Background: Protein phosphatase type 2A (PP2A) is an essential protein serine/threonine phosphatase that is conserved in all eukaryotes. PP2A is a key enzyme within various signal transduction pathways as it regulates fundamental cellular activities such as DNA replication, transcription, translation, metabolism, cell cycle progression, cell division, apoptosis and development (1-3). The core enzyme consists of catalytic C and regulatory A (or PR65) subunits, with each subunit represented by α and β isoforms (1). Additional regulatory subunits belong to four different families of unrelated proteins. Both the B (or PR55) and B' regulatory protein families contain α, β, γ and δ isoforms, with the B' family also including an ε protein. B'' family proteins include PR72, PR130, PR59 and PR48 isoforms, while striatin (PR110) and SG2NA (PR93) are both members of the B''' regulatory protein family. These B subunits competitively bind to a shared binding site on the core A subunit (1). This variable array of holoenzyme components, particularly regulatory B subunits, allows PP2A to act in a diverse set of functions. PP2A function is regulated by expression, localization, holoenzyme composition and post-translational modification. Phosphorylation of PP2A at Tyr307 by Src occurs in response to EGF or insulin and results in a substantial reduction of PP2A activity (4). Reversible methylation on the carboxyl group of Leu309 of PP2A has been observed (5,6). Methylation alters the conformation of PP2A, as well as its localization and association with B regulatory subunits (6-8).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: Protein phosphatase type 2A (PP2A) is an essential protein serine/threonine phosphatase that is conserved in all eukaryotes. PP2A is a key enzyme within various signal transduction pathways as it regulates fundamental cellular activities such as DNA replication, transcription, translation, metabolism, cell cycle progression, cell division, apoptosis and development (1-3). The core enzyme consists of catalytic C and regulatory A (or PR65) subunits, with each subunit represented by α and β isoforms (1). Additional regulatory subunits belong to four different families of unrelated proteins. Both the B (or PR55) and B' regulatory protein families contain α, β, γ and δ isoforms, with the B' family also including an ε protein. B'' family proteins include PR72, PR130, PR59 and PR48 isoforms, while striatin (PR110) and SG2NA (PR93) are both members of the B''' regulatory protein family. These B subunits competitively bind to a shared binding site on the core A subunit (1). This variable array of holoenzyme components, particularly regulatory B subunits, allows PP2A to act in a diverse set of functions. PP2A function is regulated by expression, localization, holoenzyme composition and post-translational modification. Phosphorylation of PP2A at Tyr307 by Src occurs in response to EGF or insulin and results in a substantial reduction of PP2A activity (4). Reversible methylation on the carboxyl group of Leu309 of PP2A has been observed (5,6). Methylation alters the conformation of PP2A, as well as its localization and association with B regulatory subunits (6-8).