Anti rabbit secondary antibody

Western blotting was performed as described previously.25 (link) Briefly, proteins were transferred to nitrocellulose membrane using a Trans-Blot Turbo Transfer System (BioRad, Montreal, QC, Canada), transfer was confirmed using Ponceau S stain (0.1% (w/v) in 5% (v/v) acetic acid) and the membranes were blocked in 5% skim milk in Tris-buffered saline with 0.1% (v/v) Tween-20 (TBS-T; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20) at room temperature for 2 hours. The membranes were incubated overnight at 4°C with the following primary antibodies diluted in 5% (w/v) skim milk in TBS-T: anti-poly-(ADP- ribose) polymerase (PARP) (1:3,000, 9542S; Cell Signaling Technology, Whitby, ON, Canada), anti-caspase 3 (1:500, 9662S; Cell Signaling Technology), anti-caspase 7 (1:1,000, 9492S; Cell Signaling Technology), anti-caspase 9 (1:1,000, 9502S; Cell Signaling Technology), and anti-β-actin (1:5,000, 8457L; Cell Signaling Technology). Membranes were washed three times with TBS-T, incubated with anti- rabbit secondary antibody (1:3,000, 7074S, Cell Signaling Technology), washed three times with TBS-T, incubated with Western Lightning Plus-ECL reagent (PerkinElmer Inc., Waltham, MA, USA) for 2 minutes, and developed using the chemiluminescence setting on a ChemiDoc MP Imaging System (BioRad).

Expression of phosphoproteins for the samples of all the subjects (n = 10) were evaluated using anti-phospho-Forkhead box protein O1 (FOXO1^Ser256, 1:1000, sc-101681), anti-phospho-AMP-activated protein kinase (AMPKα1/2^Thr172, 1:200; sc-33524, all from Santa Cruz Biotechnology, USA), anti-phospho-p38 mitogen- activated protein kinase (p38 MAPK^{Thr180/Tyr182}, 1:500; ab4822), anti-phospho-Ca²⁺/calmodulin-dependent protein kinase (CaMKII^Thr286, 1:2500; ab32678) or anti-GAPDH (1:2500; ab9485) antibodies (the latter three were all from Abcam, UK), and anti-rabbit secondary antibody (Cell Signaling, USA) as described elsewhere [29 (link)]. Luminescent signals were captured using a ChemiDoc imaging system (Bio-Rad, USA). All data are expressed as the ratio of target protein to GAPDH.

Cells were lysed in RIPA buffer (Sigma-Aldrich; Merck KGaA). The protein concentration in the samples was determined using a Bradford protein assay (Bio-Rad Laboratories, Inc.). Equal amounts of cell lysate (30 µg/lane) were separated via 10% polyacrylamide gel electrophoresis, and transferred onto PVDF membranes, which were blocked using 5% w/v non-fat milk containing TBS-Tween 20 (TBST; 0.05% v/v) for 1 h at room temperature. The membranes were incubated with antibodies against DCTN2 (cat. no. D122213; Sangon Biotech Co., Ltd., Shanghai, China) and ROCK1 (cat. no. 4035; Cell Signaling Technology, Inc.) overnight at 4°C, washed with TBST, and then incubated with anti-rabbit secondary antibody (cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 1 h. β-actin (cat. no. D191047; Sangon Biotech Co., Ltd.; 1:1,000) was detected as a reference protein for equal sample loading. ECL reagents (cat. no. 32106; Thermo Fisher Scientific, Inc.) were used to visualize the protein bands, which were captured on X-ray films. Image Lab 5.0 software (Bio-Rad Laboratories, Inc.) was used for quantitative analysis.

Propidium iodide (PI), PBS, and RPMI1640 medium were purchased from Thermo Fisher Scientific. DMSO and Hoechst 33 342 (Ho) were purchased from Sigma Aldrich. Anti–poly ADP‐ribose polymerase 1 (PARP) antibody, anti–cleaved caspase‐3 antibody, anti–γ‐H2A.X (ser139) antibody, anti–mutS homolog 2 (MSH2) antibody, anti–mutS homolog 6 (MSH6) antibody, anti–mutL homolog 1 (MLH1) antibody, anti–PMS2 antibody, anti–RAD23 homolog A (RAD23A) antibody, anti–Fanconi anemia group D2 (FANCD2) antibody, anti–O⁶‐methylguanine‐DNA methyltransferase (MGMT) antibody, and anti–rabbit secondary antibody were purchased from Cell Signaling Technology. Anti–dynein cytoplasmic 2 heavy chain 1 (DYNC2H1) antibody was purchased from Abcam, TMZ and ACNU (dissolved in DMSO) were purchased from Fujifilm Wako Pure Chemical Corporation, and CCNU (dissolved in DMSO) was purchased from Tokyo Chemical Industry.