Anti actin mab a2228

Cell lysates were extracted with Ripa lysis buffer (50mM Tris, pH 7.4, 150mM NaCl, 1% Triton X-100,1% sodium deoxycholate,0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA and leupeptin) supplemented with Roche Complete protease inhibitor cocktail. The protein concentration in the supernatant was measured with a bicinchoninic acid (BCA) assay kit (Thermo). SDS-PAGE was then performed using a 10% or 12% gel to isolate the proteins. After electrophoresis, proteins were transferred onto nitrocellulose filter (NC) membrane (Whatman) and hybridized with primary antibodies. The primary antibodies anti-TBP (T1827) and anti-actin mAb (A2228) were from Sigma-Aldrich (Shanghai, China). Anti-p65 antibody anti-IKKα, and anti-IKKβ were from Cell Signaling Technology (#9936, Danvers, MA, USA). The Page Ruler Prestained Protein Ladder from Thermo Scientific (#26616, Waltham, MA, USA) was used to indicate the target protein. Detection and quantification were performed with the Li-cor Odyssey imaging system and its software (Lincoln, NE, USA). 1:1000 overnight at 4°C. The secondary antibodies (IRdey800/680 goat anti mouse, or IRdey800/680 goat anti rabbit, Li-Cor) were all used at a dilution of 1:10000.

Co-IP and IP were performed as described.^{31 (link)} The primary antibodies anti-TBP (T1827) and anti-actin mAb (A2228) were from Sigma-Aldrich (Shanghai, China). Anti-p65 (#4764), anti-IκBα (#4814s) and anti-cleaved caspase-3 (#9661) antibody were from Cell Signaling Tech (Danvers, MA, USA). Anti-myc (9E10) was from Abcam (Cat: ab32, Mouse monoclonal, Cambridge, MA, USA). Anti-IκBα (phospho-Y42) (Rabbit polyclonal, Cat: BS4736,) and anti-IκBα (phospho-S32/S36) (Rabbit polyclonal, Cat: BS4105) were from Bioworld Tech (St. Louis Park, MN, USA). The PageRuler Prestained Protein Ladder from Thermo Scientific (#26616, Waltham, MA, USA) was used to indicate the target protein. Detection and quantification were performed with the Li-cor Odyssey imaging system and its software (Lincoln, NE, USA).