Anti phospho nf kb p65

Cell lines and primary cells were treated with acalabrutinib and stimulated with plate-bound anti-human IgM (MP Biomedicals; Santa Ana, CA). Plates were prepared by incubating a 10 μg/mL IgM solution in PBS for 6 to 12 hours at 4°C, and then rinsing with PBS. Whole cell lysates were prepared as previously described [24 (link)], followed by polyacrylamide gel electrophoresis and transfer of proteins to nitrocellulose membranes. The following polyclonal antibodies were used to detect protein on immunoblots: anti-phospho-PLCG2 (Tyr 1217, Cat. #3871), anti-PLCG2 (Cat. #3872), anti-phospho-IKBA (Ser32, Cat. #2859), anti-IKBA (Cat. #4812), anti-phospho-ERK1/2 (Thr202/Tyr204, Cat. #9101), anti-ERK1/2 (Cat. #9102), anti-phospho-AKT (Thr308, Cat. #9257), and anti-AKT (Cat. #9272), anti-phospho-NFKB P65 (Ser536, Cat. #3031), anti-NFKB P65 (Cat. #3034)(Cell Signaling Technologies; Danvers, MA), anti-phospho-BTK (Tyr223, Cat. #ab68217, Abcam, Cambridge, MA), and anti-BTK (cat. #B3187, Sigma-Aldrich).

Muscles were dissected, minced, and homogenized in 0.5 mL RIPA buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5 M NaCl, 0.5% NaDoc, and 1% NP40) supplemented with Protease Inhibitor Cocktails (Roche 11697498001 and 04906837001, Germany) using a Dounce tight pestle. The homogenate was passed through a 16G needle. Proteins (100 μg) were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membrane (Amersham Piscataway, NJ). Nonspecific binding was blocked in Tris-Cl Buffered Saline Solution with 0.05% Tween-20 (TBST) containing 10% nonfat dry milk (Nestlé) Blocking Buffer (BB) overnight at +4°C and then probed 1 h with primary antibody. The following specific antibodies were used: 

Anti-Pax7 1 : 50 in BB (Hybridoma supernatant, Iowa University, IO).

 

Anti-MyoD 1 : 50 in TBST (Santa Cruz).

 

Anti-Desmin 1 : 50 IN BB (Sigma-Aldrich, Saint Louis, MO).

 

Anti-phospho-NF-kB: p65 1 : 400 in 5% BSA (Cell Signaling, 3033).

 

Anti-NF-kB p65 1 : 400 in 5% BSA (Cell Signaling, 4764).

 

Anti-GAPDH 1 : 10000 (Santa Cruz).

After washing in TBST, blots were incubated with anti-mouse or anti-rabbit secondary antibody HRP-conjugated (BioRad, Hercules, CA) diluted 1 : 10000 in TBST and detected by using Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Cells were harvested using a centrifuge and lysed in PRO-PREP solution (iNtRON Biotechnology, Seongnam, Republic of Korea, 17081). Total protein levels were quantified using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA, 23225). Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA, 66485). After blocking with 5% (w/v) skim milk (MB cell, Seoul, Republic of Korea, MB-S1667), membranes were incubated with primary antibodies at 4 °C overnight. After washing off unbound primary antibodies, membranes were incubated with peroxidase-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence system (TransLab, Daejeon, Republic of Korea, TLP-112.1) after incubation at room temperature for 2 h. The following primary antibodies were used in this study: anti-WWOX (Abcam, ab28144), anti-phospho-NF-kB p65 (Cell Signaling Technology, Danvers, MA, USA, #3033), and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-47778). Protein bands were quantified by ImageJ software v1.53a (National Institutes of Health).