Poly adp ribose polymerase parp

Equal amounts of proteins were separated by SDS-PAGE, and then transferred onto polyvinylidene membranes (Millipore, SaintQuentin enYvelines, Belgium) by electrotransfer. Membranes were blocked with 5% skim milk in PBS-T (containing 0.1% Tween-20), and proteins of interest were visualized using specific Anti-Stathmin (Cell SignalingTechnology, Beverly, MA, USA), poly (ADP-ribose) polymerase (PARP) (BD Biosciences, San Jose, CA, U.S.A.), integrin β1, and integrin α5(BD Biosciences, San Jose, CA), caspase-3 (Santa CruzBiotech, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody and secondary antibodies, conjugated with horseradish peroxidase (HRP), were ordered from KangChen Biotech (Shanghai, China).
For immunoprecipitation, the cells were lysed inbuffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet™ P-40 (NP-40), 5 mM EDTA, 5 mM ethylene glycol tetraacetic acid, 15 mM MgCl2, 60 mM β-glycerolphosphate, 0.1 mM sodiumorthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF. Twenty microliters of protein A/Gagarose beads (BD Bioscience Pharmingen) were added to the lysates for proper periods of incubation. The beads were then washed and subjected to SDS-PAGE and immunoblotting.

Cells were grown to ∼70% confluence and reagents were added as indicated. Western blot analysis was performed as described previously (36 (link),37 (link)) with the following antibodies: phospho-SRC (Y416), phospho-STAT3 (T705), and SRC (Cell Signaling Technology, Inc., Boston, MA, USA), ALDH1A1 (Abcam, Cambridge, UK), Poly-ADP-ribose-polymerase (PARP; BD Biosciences, Franklin, NJ, USA), α-tubulin and β-actin (Sigma, St. Louis, MO, USA).

Ibrutinib was purchased from Selleckchem (Houston, TX, USA), cerdulatinib was provided by Portola Pharmaceuticals Inc. (South San Francisco, CA, USA), CpG (ODN2006, stimulatory CpG-ODN type B, human specific) was purchased from Invivogen (San Diego, CA, USA), IL-6 was from R&D Systems (Minneapolis, MN), and IL-4 and CD40L were from Enzo Life Sciences (Plymouth Meeting, PA, USA). Antibodies: anti-phosphorylated BTK (p-BTK) (Y223), p-IκBα (S32/36) p-STAT3 (Y705), STAT3, MCL-1, p-JAK1 (Y1022), p-JAK3 (Y980), p-STAT6 (Y641), p-STAT3 (S727), and p-JAK2 (T1007/1008) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-total BTK antibody, poly ADP-ribose polymerase (PARP), and the BrdU detection kit were from BD Biosciences (San Jose, CA, USA); anti-p65, STAT-6, STAT-3, JAK1, JAK2, JAK3 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For flow cytometry, FITC-anti-CD19 (clone HIB19) and PE-anti-CD5 (clone UCHT2) were purchased from eBioscience (San Diego, CA, USA). Alexa Fluor® 647-anti-p-AKT (Ser473) and Alexa Fluor® 488 anti-p-p44/42 MAPK (ERK1/2) (T202/Y204) were purchased from Cell Signaling Technology, and PE-anti-p-PLCγ2 (Y759) was purchased from BD Bioscience.