Western blot analysis of extracts from BaF3 cells starved of mouse IL-3 and serum overnight, untreated (-) or mouse IL-3-treated (10 ng/ml, 6 hr; +) and NK-92 cells starved of human IL-2 and serum overnight, untreated (-) or human IL-2-treated (10 ng/ml, 6 hr; +), using SOCS1 (A156) Antibody.
Western blot analysis of extracts from K562, A204 and PC12 cells using SOCS2 Antibody.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human SOCS3 (+), using SOCS3 (D6E1T) Rabbit mAb.
Flow cytometric analysis of HeLa cells using PIAS1 (D33A7) XP® Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
Confocal immunofluorescent analysis of A-204 (positive, left) and Hep G2 (low expression, right) cells using PIAS3 (D5F9) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Western blot analysis of extracts from 293 and CCRF-CEM cells using PIAS4 (D2F12) Rabbit mAb.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from COS-7 cells, untransfected (-) or transfected with a construct overexpressing human SOCS1 (+), using SOCS1 (A156) Antibody.
Western blot analysis of extracts from Hep G2 and A-375 cell lines, untreated (-) or treated with Human Oncostatin M #5367 (hOSM; 20 ng/ml, 1 hr; +), using SOCS3 (D6E1T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Confocal imunofluorescent analysis of HeLa cells using PIAS1 (D33A7) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
Western blot analysis of extracts from RD cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® PIAS3 siRNA I #9073 (+), or SignalSilence® PIAS3 siRNA II #9031 (+), using PIAS3 (D5F9) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The PIAS3 (D5F9) XP® Rabbit mAb confirms silencing of PIAS3 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
Western blot analysis of extracts from KARPAS-299 cells, untreated (-) or treated with the Jak3 inhibitor WHI-P154 (40 μM, overnight; +), using SOCS3 (D6E1T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb (lower). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
Western blot analysis of extracts from various cell lines using PIAS1 (D33A7) XP® Rabbit mAb.
Western blot analysis of extracts from various cell lines using PIAS3 (D5F9) XP® Rabbit mAb.
Western blot analysis of extracts from mouse bone marrow derived macrophage (mBMDM) cell extracts, untreated (-) or treated with Lipopolysaccharides #14011 (LPS; 50 ng/ml, 4 hr; +), using SOCS3 (D6E1T) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human PIAS3 (hPIAS3; +), using PIAS3 (D5F9) XP® Rabbit mAb.
|SOCS1 (A156) Antibody 3950||20 µl||
||H Mk M R||23||Rabbit|
|SOCS2 Antibody 2779||20 µl||
||H M R||22||Rabbit|
|SOCS3 (D6E1T) Rabbit mAb 52113||20 µl||
||H M||28||Rabbit IgG|
|PIAS1 (D33A7) XP® Rabbit mAb 3550||20 µl||
||H Mk M R||76||Rabbit IgG|
|PIAS3 (D5F9) XP® Rabbit mAb 9042||20 µl||
|PIAS4 (D2F12) Rabbit mAb 4392||20 µl||
||H Mk R||75||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Jak/Stat Pathway Inhibitors Antibody Sampler Kit provides an economical means to examine several inhibitors of Jak/Stat signaling, including PIAS1, PIAS3, PIAS4, SOCS1, SOCS2, and SOCS3. The kit contains enough primary antibody to perform two western blot experiments with each primary antibody.
Each antibody in this kit recognizes only the specific target protein and does not cross-react with other family members.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala156 of human SOCS1 protein, residues at the carboxy terminus of human SOCS2 protein, or recombinant protein specific to human SOCS3 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser550 of human PIAS1 protein, Pro590 of human PIAS3 protein, or Lys59 of human PIAS4 protein.
Jak (Janus Kinase) and Stat (signal transducer and activator of transcription) proteins are utilized by receptors for a wide varity of ligands including cytokines, hormones, growth factors, and neurotransmitters (1). Jaks and Stats play important roles in oncogenesis, tumor progression, angiogenesis, cell motility, immune responses, and stem cell differentiation (2-5). Therefore, regulation of Jak/Stat signaling is crucial to prevent aberrant signaling which can lead to disease progression. Two methods for regulating Jak/Stat signaling involve SOCS and PIAS proteins (6,7).
The SOCS (suppressor or cytokine signaling) family members are negative regulators of cytokine signal transduction that inhibit the Jak/Stat pathway and consist of 8 known members, including the originally identified protein CIS1 (cytokine-inducible SH2-containing protein) and SOCS1-SOCS7. Each SOCS family member contains a central SH2 domain and a conserved carboxy-terminal motif designated as the SOCS box. These proteins are important regulators of cytokine signaling, proliferation, differentiation, and immune responses (8-10). SOCS proteins are involved in regulating over 30 cytokines, including interleukins, growth hormone (GH), interferons, leptin, and leukemia inhibitory factor (7). SOCS1, also known as JAB (Janus Kinase binding protein) and SSI-1 (Stat-induced Stat inhibitor-1), shares the most homology with SOCS3 and both are highly induced by cytokines (7,11). Both SOCS1 and SOCS3 directly inhibit Jak activity; SOCS1 inhibits Jak through an interaction involving a phospohotyrosine located in the kinase activation loop; SOCS3 inhibits Jak via its SH2 domain (12,13). In addition to inhibiting Jak/Stat signaling, the SOCS box of SOCS1 and SOCS3 can trigger ubiquitin-mediated degradation of proteins within and outside the Jak/Stat pathway (14,15). SOCS2 is also incduced upon cytokine stimulation and the activity of SOCS2 has been predominately linked to GH and insulin-like growth factor signaling by binding to tyrosine-phosphorylated receptors via its SH2 domain (11,16).
The PIAS (protein inhibitor of activated Stats) proteins, which include PIAS1, PIAS3, PIASx, and PIASy (PIAS4), were originally characterized based on their interaction with the Stat family of transcription factors (16,17). PIAS1, PIAS3, and PIASx interact with and repress Stat1, Stat3, and Stat4, respectively (17-19). The PIAS family contains a conserved RING domain that has been linked to function as a SUMO (small ubiquitin-related modifer) ligase, coupling the SUMO conjugating enzyme Ubc9 with its substrate proteins leading to regulation of transcription factors through distinct mechanisms including NF-κB, c-Jun, Oct-4, p53, and SMADs. PIAS4 is a specific SUMO-E3 ligase for Ets-1 and represses Ets-1 dependent transcription in addition to altering the nuclear localization and reducing the transcriptional activity of C/EBPδ, thereby enhancing cell proliferation and migration (20,21).
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