|H M R Mk||Endogenous||22, 28||Rabbit IgG|
Western blot analysis of extracts from various cell lines using PSMB5 (D1H6B) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 72 hr; +), using PSMB5 (D1H6B) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human PSMB5 (hPSMB5-Myc/DDK; +) or Myc/DDK-tagged full-length human PSMB8 (hPSMB8-Myc/DDK; +), using PSMB5 (D1H6B) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
PSMB5 (D1H6B) Rabbit mAb recognizes endogenous levels of total PSMB5 protein. This antibody reacts with precursor and mature forms of PSMB5. This antibody does not cross-react with PSMB8.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PSMB5 protein.
The 26S proteasome is a highly abundant proteolytic complex involved in the degradation of ubiquitinated substrate proteins. It consists largely of two sub-complexes, the 20S catalytic core particle (CP) and the 19S/PA700 regulatory particle (RP) that can cap either end of the CP. The CP consists of two stacked heteroheptameric β-rings (β1-7) that contain three catalytic β-subunits and are flanked on either side by two heteroheptameric α-rings (α1-7). The RP includes a base and a lid, each having multiple subunits. The base, in part, is composed of a heterohexameric ring of ATPase subunits belonging to the AAA (ATPases Associated with diverse cellular Activities) family. The ATPase subunits function to unfold the substrate and open the gate formed by the α-subunits, thus exposing the unfolded substrate to the catalytic β-subunits. The lid consists of ubiquitin receptors and DUBs that function in recruitment of ubiquitinated substrates and modification of ubiquitin chain topology (1,2). Other modulators of proteasome activity, such as PA28/11S REG, can also bind to the end of the 20S CP and activate it (1,2).
The core particle performs three types of catalytic activities inside its chamber: chymotrypsin-like, trypsin-like, and caspase-like activities, which are provided by the constitutively expressed PSMB5 (β5/MB1/X/LMPX/Macropain epsilon chain), PSMB7 (β2/Z/Macropain chain Z) and PSMB6 (β1/Y/LMPY/Macropain delta chain) subunits, respectively. These catalytic subunits belong to the amino-terminal nucleophile (Ntn) hydrolase family and are characterized by a single-residue active site. The catalytic β-subunits are synthesized with amino-terminal propeptides, which are removed at the final step of proteasome biogenesis to expose the catalytic threonine residues (3). In immune cells involved in antigen presentation, the constitutively expressed PSMB6, PSMB7, and PSMB5 subunits are replaced by three highly homologous induced β-subunits: PSMB9 (β1i/LMP2/RING12), PSMB10 (β2i/MECL-1/LMP10) and PSMB8 (β5i/LMP7/RING10), respectively, to form the immunoproteasome (4,5). PSMB5 is downregulated at the protein level by IFN-γ and replaced by PSMB8 in order to remodel the proteolytic specificity of the proteasome for more appropriate immunological processing of endogenous antigens (6-8). PSMB5 is also one of the predominant targets of bortezomib, an inhibitor of the chymotrypsin-like activity of the proteasome (9).
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