Western blot analysis of extracts from various cell lines using PSMB8/LMP7 (D1K7X) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). (The T2 cell line contains a homozygous deletion of PSMB8/LMP7 (13)).
Western blot analysis of extracts from HeLa and SW620 cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 72 hr; +), using PSMB8/LMP7 (D1K7X) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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 PSMB8/LMP7 (D1K7X) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).
|REACTIVITY||H M R|
|MW (kDa)||23, 28|
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.
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
PSMB8/LMP7 (D1K7X) Rabbit mAb recognizes endogenous levels of total PSMB8/LMP7 protein. This antibody recognizes both 28 kDa precursor and 23 kDa mature forms of PSMB8/LMP7 and does not cross-react with PSMB5 protein. This antibody recognizes proteins of unknown origin in the 80-100 kDa range.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PSMB8/LMP7 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).
Constitutively expressed core particle subunits PSMB5, PSMB7, and PSMB6 provide chymotrypsin-like, trypsin-like, and caspase-like activities, respectively (3). In immune cells involved in antigen presentation, these subunits are replaced by highly homologous, induced β-subunits to form the immunoproteasome (4,5).
Proteasome subunit beta type-8 (PSMB8, LMP7) is expressed as a proenzyme that is cleaved to form the mature PSMB8 (LMP7) immunoproteasome core particle subunit (6). Interferon-γ induces expression of PSMB8, which functionally replaces the PSMB5 core particle subunit in immunoproteasome processing of MHC class I-restricted peptide antigens (7). Research studies suggest that reduced PSMB8 expression or expression of the non-functional LMP7-E1 isoform may impair immunoproteasome assembly, and that PSMB8 deficiency results in reduced MHC class I molecule expression (8-10). Inhibition of PSMB8 in murine rheumatoid arthritis models attenuates disease indicators, suggesting that PSMB8 is a potential therapeutic target in the treatment of some proinflammatory autoimmune diseases (11). Mutations in the corresponding PSMB8 gene can cause an autoinflammatory syndrome known as CANDLE Syndrome (12).
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