Ab179461

Cells were lysed by the RIPA lysis buffer (Beyotime, China) on ice. Protein concentrations were quantified by BCA protein assay (Beyotime, China). The immunoblots were probed with appropriate primary overnight at 4°C followed by incubation with the corresponding secondary antibodies at room temperature for 1 h.
The blots were infiltrated with ECL (Bio-Rad, USA) and detected by ChemiDoc™MP Imaging System (Bio-Rad, USA). The densitometry of target bands was normalized to internal control β-actin. The antibodies are listed as follow up: Runx2 (1:1000, ET1612-47, Huabio, Hangzhou, China); OPN (1:1000, 0806–6, Huabio, Hangzhou, China); cleaved-caspase 3 (1:1000, 9664S, Cell Signaling Technology, USA); cleaved-caspase 9 (1:1000, 9509S, Cell Signaling Technology, USA); TOM20 (1:1000, 42406S, Cell Signaling Technology, USA); cytochrome c (1:1000, 12,963, Cell Signaling Technology, USA); T-JNK (1:1000, ab179461, Abcam, UK); p-JNK (1:1000, ab124956, Abcam, UK); T-c-JUN (1:1000; ET1608-3, Huabio, Hangzhou, China); p-c-JUN (1:1000; ET-1608-4, Huabio, Hangzhou, China); β-actin (1:3000, R1102-1, Huabio, Hangzhou, China); tubulin (1:3000, M1305-2, Huabio, Hangzhou, China); anti-rabbit, anti-mouse secondary antibody (1:3000, #HA1011, #HA1006, Huabio, Hangzhou, China).

Cells were washed with 37 °C PBS, mixed with RIPA Lysis Buffer for 10 min at 4 °C, and centrifuged to yield whole-cell lysates. The protein sample (20 µg) was separated by SDS polyacrylamide gels with electrophoresis (Bio-Rad, USA), and the gel was transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer for 2 h and then incubated overnight at 4 °C with the corresponding primary antibodies, including phospho-extracellular signal-regulated kinase (P-ERK) (1:1,000 dilution, #4370, CST, USA), ERK (1:1,000 dilution, #4695, CST, USA), phospho-c-Jun N-terminal kinase (P-JNK) (1:1,000 dilution, ab124956, Abcam, Cambridge, UK), JNK (1:1,000 dilution, ab179461, Abcam, Cambridge, UK), phospho-p38 mitogen-activated protein kinase (P-p38 MAPK) (1:1,000 dilution, #9211, CST, USA), p38 MAPK (1:1,000 dilution, #9212, CST, USA), NP (1:1,000 dilution, bs-4976R, Bioss, Beijing, China), and GAPDH (1:700, M00227-5, Boster, Wuhan, China). The membrane was washed with TBS-T and incubated with the HRP-goat anti-Rabbit IgG (1:5000 dilution, BA1054, Boster, Wuhan, China) as the secondary antibody and then developed in electrochemiluminescent western detection reagents (Millipore, Billerica, MA, USA).