Anti p jnk

The following antibodies were used for immunoblotting: Cell Signaling Technology, anti-p38 (Cat#: 8690), anti-p-p38 (Cat#: 4511), anti-p65 (Cat#: 8242), anti-p-p65 (Cat#: 3033), anti-JNK (Cat#: 9252), anti-p-JNK (Cat#: 4668), anti-Erk (Cat#: 4695), anti-p-Erk (Cat#: 4370), anti-PKM2 (Cat#: 4053), anti-STAT3 (Cat#: 12640); Abcam, anti-Pyk2 (Cat#: 228477); Santa Cruz Biotechnology, anti-p-Pyk2 (Cat#: sc-81512); Beyotime Institute of Biotechnology, anti-β-actin (Cat#: AA128), HRP labeled Goat Anti-Rabbit IgG (Cat#: A0208), HRP-labeled Goat Anti-Mouse IgG (Cat#: A0216). The following antibodies purchased from Biolegend were used for flow cytometry: FITC anti-mouse B220 (Cat#: 103206), BV421 anti-mouse CD11c (Cat#: 117330), FITC anti-mouse F4/80 (Cat#: 123108), APC anti-mouse CD86 (Cat#: 105012), PE anti-mouse CD40 (Cat#: 124610), PE anti-mouse GL7 (Cat#: 144607), APC anti-mouse CD95 (Cat#: 152604), APC anti-mouse CXCR5 (Cat#: 145506), PE anti-mouse PD-1 (Cat#: 135206), APC/Cy7 anti-human CD19 (Cat#: 302218), FITC anti-human CD14 (Cat#: 367116), BV421 anti-human CD11c (Cat#: 301628), APC anti-human CD86 (Cat#: 374208), PE anti-human CD40 (Cat#: 334308). All the antibodies for flow cytometry were used at a 1:100 dilution.

CD44⁺ BM cells harvested from transwell systems were lysed with SDS lysis buffer (100 mM Tris at pH 8.0, 10% glycerol, and 1% SDS) and protein concentration was determined using a NanoVue Spectrophotometer (GE Healthcare Life Sciences, USA). A total of 30 μg of protein lysates was separated by SDS-PAGE (80 V, 120 min; Beyotime, Shanghai, China) and transferred to polyvinylidene difluoride membranes (250 mA × 60 min, Millipore, USA). After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour. Signals were detected by enhanced chemiluminescence (KPL, Gaithersburg, MD). GAPDH was used as the loading control. Western blot was repeated 3 times for each cell batch.

About 100 mg of bladder tissue were lysed by RIPA lysate with a final concentration of 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein quantification was performed using the Bio-Rad DC Protein Assay Kit (Guangzhou Yuwei Biotechnology Instrument Co., Ltd., Guangzhou, China). Each sample was added SDS loading buffer. And boiling in water for 10 min, the sample was load on a 10% SDS–polyacrylamide gel and run at 120 V, 30 min, and 100 V for 90 min. The protein was transferred from the gel to a PVDF membrane. The membrane was immersed in 1 × TBST containing 5% skimmed milk powder and gently shaken at room temperature for 2 h to block non-specific binding sites. PVDF membrane was wash 1 × TBST 3 times, 5 min/time. Add primary antibodies (anti-CB1, rabbit, 1: 250; Anti-NF-kB p65, rabbit, 1: 1000; anti-p-JNK, rabbit, 1: 1000; Abcam), incubated at 4 °C overnight, washed 3 times with 1 × TBST, 5 min/time, and then added with secondary antibody IgG (Affinity Biosciences Bio, S0001, goat anti-rabbit, 1: 20000) respectively and incubated 3 times with 1 × TBST at room temperature for 1 h. Development was performed with ECL. The gray value of Western blotting experimental protein expression was determined by Image J software, and the experiment was repeated three times.

The protocol has been described previously [12 (link)]. Proteins of interest were detected using anti-iNOS (1:1000, Novus), anti-eNOS (1:1000, Abcam), anti-alkaline phosphatase (ALP; 1:1000, Abcam), anti-runt-related transcription factor 2 (Runx2; 1:1000, Abcam), anti-peroxisome proliferator-activated receptor (PPAR)γ (1:1000, Abcam), anti-p-JNK (1:1000, Abcam), or anti-JNK (1:1000, Abcam) antibodies, while β-actin was detected with anti-β-actin antibody (1:2000, Abcam) as a control.

MTT, dimethyl sulfoxide (DMSO), PI, DAPI and DCFH-DA were purchased from Sigma (St Louis, MO). RIPA buffer and JC-1 staining kit were purchased from Beyotime Biotechnology, China. The following antibodies were used: anti-PUMA, anti-p53, anti-Chk1, anti-Chk2, anti-p-Chk1(Ser345), anti-p-Chk2 (Thr68), anti-ATM, anti-p-ATM (Ser1981), anti-PARP, anti-GAPDH and anti-β-actin (Santa Cruz Technology, Santa Cruz, CA, USA); Anti-NOX1, anti-NOX4, anti-DUOX1 and anti-SOD1, anti-Keap1, anti-Nrf2, anti-HO-1, anti-DNA-PKcs, anti-p-DNA-PKcs (Thr 2609), anti-Caspase-3 and anti-Caspase-9 (Cell Signaling Technology, Danvers, MA, USA); Anti-γ H2AX (Ser139), anti-H2AX and anti-JNK, anti-p-JNK, anti-BCL-XL, anti-MCL-1 and anti-BCL-2 (Abcam). Rhodamine (TRITC) AffiniPure Goat anti-Rabbit IgG was from Santa Cruz Biotechnology; KU-60019, VE-821 and NU7026 were obtained from Selleck (USA). SP600125 were obtained from Calbiochem (La Jolla, CA, USA). Z-VAD-fmk was purchased from R&D Systems (Minneapolis, MN). These inhibitors were dissolved in DMSO and diluted to appropriate concentrations with cell culture media. Mitotracker and MitoSOX red mitochondrial superoxide indicator was purchased from Molecular Probe (USA).