Anti bcl 2

Cell lysates were obtained using radioimmunoprecipitation assay buffer (Beyotime, China). After microcentrifugation, the supernatant was subjected to dose assessment using a BCA protein quantitative kit (Hualikexi, China). Equal amounts of protein were loaded onto a 10% SDS-PAGE following electrophoresis. Following membrane transfer, the membrane was blocked with 5% nonfat milk for 30 min at room temperature. Anti-Bax (1:1000, cat: 33–6400, Thermo Fisher), anti-bcl-2 (1:1000, cat: MA5-11757, Thermo Fisher), anti-LAMC1 (1:1000, cat:PA5-36300, Thermo Fisher), and anti-GAPDH (1:1000, cat:MA1-16757, Thermo Fisher) antibodies were used to label the proteins on the membranes in a cool room overnight. Subsequently, secondary antibodies were used to detect the target proteins on the membranes. The Pierce ECL Western blot substrate was used for staining.

Radioimmunoprecipitation assay lysis buffer was used to prepare SW480 and SW620 cell lysates. After microcentrifugation, the protein samples were quantified using a BCA Protein Assay kit (Abcam, USA). Protein samples (20 µg) were separated using 10% SDS-PAGE and transferred to a PVDF membrane at a constant voltage of 75 V. The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated with anti-AURKB (Cat#:36-5200; 1:1,000; Thermo Fisher), anti-Bax (Cat#:33-6400; 1:1,000; Thermo Fisher), anti-Bcl-2 (Cat#: MA5-11757; 1:1,000; Thermo Fisher), and anti-GAPDH antibodies (Cat#: MA1-16757; 1:1,000; Thermo Fisher) overnight at 4°C. The next day, the membranes were incubated at room temperature with HRP-conjugated goat anti-rabbit/mouse secondary antibodies (Cat#: SA5-10204 or A32727; 1:1,000; Thermo Fisher) for 2 h. The blots were then developed using BeyoECL Plus mix substrate (Beyotime, USA), and ImageJ (Image J Software, National Institutes of Health, USA) was used to quantify the target proteins.

Total proteins were extracted by RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China). The protein concentration was measured by the BCA Protein Assay Kit (Thermo Fisher, Waltham, USA). Then, equal amounts of protein were loaded on 8–12% SDS-PAGE gels and transferred onto a PVDF transfer membrane (Millipore, Massachusetts, USA). After blocking with 5% skimmed milk solution, the membranes were incubated with specific primary antibodies overnight at 4°C followed by further incubation with the secondary antibody at room temperature for 2 h. Both primary and secondary antibodies were purchased from Thermo Fisher (Waltham, USA), including anti-β-actin, anti-Nrf2, anti-HO-1, anti-p-NF-κB, anti-IL-1β, anti-TNF-α, anti-Bip, anti-calpain-2, anti-active caspase-12, anti-active caspase-3, anti-Bax, anti-Bcl-2, anti-ACAN, anti-Col-II, anti-Col-I, anti-MMP-13, anti-aggrecanase 1, and secondary antibody IgG. Finally, the protein bands were detected using the ECL Plus Reagent (Thermo Fisher, Waltham, USA), and their densitometry was analyzed with the ImageJ software version 8.0(Bio-Rad, Hercules, USA). With β-actin as control, the expression of total protein was normalized.

Small molecule BDA-366 was received from the Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (NCI, Bethesda, MD). AffiniPureF(ab’)2FragmentGoatAnti-HumanIgG + IgM (H + L) (IgG/IgM) was purchased from SANBIO (CA, USA).
Immunoblotting was performed on DLBCL with anti-vinculin (#V-9131,Sigma-Aldrich, Munich, Germany), anti-GAPDH (#G8795, Sigma-Aldrich, Munich, Germany), anti-Bax (6A7) (#Ab00120-1.1, Absolute Antibody, Oxford, United Kingdom), anti-Bcl-2 (#PA5-20068, Thermo Scientific, Brussels, Belgium), anti-Bax (#2772, Cell Signaling, MA, USA), anti-Mcl-1 (# 4572, Cell Signaling, MA, USA).
Immunoblotting of primary CLL patient samples was performed using the following antibodies: anti-PARP (#9542, Cell Signaling), anti-phospho-Bcl2 (Ser70) (#2827, Cell Signaling), anti-phospho AKT (Ser473) (#9271, Cell Signaling), anti-phospho-GSK3α/β (Ser21/9) (#9331, Cell Signaling), anti-Bim (#2819, Cell Signaling), anti-Bcl-2 (#2872, Cell Signaling), anti-β-actin (#3700, Cell Signaling), anti-Mcl-1 (#sc-819 Santa Cruz Biotechnology), anti-Bcl-xL (#sc-1041, Santa Cruz Biotechnology), anti-Bax (#sc-493, Santa Cruz Biotechnology), rabbit IgG-HRP-linked (# 111-035-046, Jackson ImmunoResearch), and mouse IgG-HRP-linked (#115-035-07, Jackson ImmunoResearch).

The total protein was extracted by RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific). Next, we rinsed the beads with 100 μL iced buffer, added 100 μL antibody-binding buffer to revolve the antibody and magnetic beads for 30 min, and then rinsed the beads 3 times using 200 μL buffer for 5 min each time. Cell lysates and antibody-bound magnetic beads were incubated for 1 h at room temperature and washed using 200 μL buffer for 5 min each time. 20 μL eluent was used to rinse the beads once and the supernatant was removed. The cell lysates were extracted for Co-IP with anti-BCL2 (1:100), Beclin1 (1:100), BAX (1:100) antibodies (Thermo Fisher Scientific), and then, precipitates were detected using western blotting with anti-Beclin1 (1:1000), BAX (1:1000), BCL2 (1:1000) antibodies, respectively.

The collected samples were lysed by Lysis Buffer (Thermo Fisher Scientific). After measure of protein concentrations with BCA Protein Assay Kit (Beyotime, Shanghai, China), an aliquot of 20 μg protein was loaded on bis-tris-acrylamide gels. The protein sample was transferred onto a PVDF membrane by using Bio-Rad blot apparatus. The membranes were incubated with primary antibodies and secondary antibodies (#31460/61-6520; 1:10,000; Thermo Fisher Scientific) in order, and exposed to a Hyperfilm ECL to develop protein blots. Abcam (Cambridge, MA, USA) provided all antibodies. The primary antibodies included anti-Bcl-2 (#MA5-11757; 1:50; Thermo Fisher Scientific), anti-Bax (#MA5-14003; 1:100; Thermo Fisher Scientific), anti-Cleaved-caspase 3 (#PA5-114687; 1:1,000; Thermo Fisher Scientific), anti-KDM1A (#PA5-17361; 1:1,000; Thermo Fisher Scientific), and anti-β-actin (#MA1-140; 1:8,000; Thermo Fisher Scientific).