Anti jnk1

30 μg of protein lysates from hUCMSCs at days 0, 5, 9, 13, 17, and 21 were injected to 10% SDS-PAGE and transferred to PVDF membranes (Merck Millipore, USA), respectively. After blocking with 3% bovine serum albumin (BSA; Sigma-Aldrich, USA), membranes were probed with anti-phospho-ERK1/2 (1 : 500, Santa Cruz, CA, USA), anti-ERK1/2 (1 : 400, BIOSS, China), anti-phospho-JNK1 (1 : 500, Santa Cruz, CA, USA), anti-JNK1 (1 : 500, Santa Cruz, CA, USA), anti-phospho-p38 (1 : 100, Santa Cruz, CA, USA), anti-p38 (1 : 250, Santa Cruz, CA, USA), or anti-GAPDH (1 : 500, Santa Cruz, CA, USA) antibodies at 4°C overnight. After being washed three times with Tris Buffered Saline with Tween® 20 (TBST), the membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (Donkey anti-Rabbit, 1 : 50,000, Abcam, USA, or Donkey anti-Mouse, 1 : 20000, Abcam, USA) at 25°C for 1 hour and developed with commercially available enhanced chemiluminescence reagent (Pioneer, China). Band intensities were determined using ImageJ software.

Western blot analyses were performed as described in [27 (link)]. The antibodies were anti-DUSP1, anti-p38MAPK, anti-JNK1, and anti-ERK2 (Santa Cruz Biotechnology, Heidelberg, Germany); anti-phospho-p38MAPK (pp38MAPK), anti-phospho-ERK (pERK), and anti-Snail (Cell Signalling Technology, Izasa S.A., Barcelona, Spain); anti-phospho-pJNK (pJNK) (Promega Biotech Ibérica, Madrid, Spain); anti-Tubulin (Sigma Aldrich, Madrid, Spain); peroxidase-conjugated secondary antibodies (GE Healthcare Europe GMBH, Barcelona, Spain). Tubulin was utilized as a loading control for Western blotting analysis. Relative protein levels compared to tubulin were analyzed by Image J software and plotted.
Immunofluorescence staining was performed as previously described [28 (link)]. Briefly, cells cultured on coverslips were fixed, permeabilized, blocked and, after several washes, stained for Snail with the specific antibody, followed by the anti-rabbit Alexa Fluor^® 488 secondary antibody (BD Biosciences, Franklin Lakes, NJ, USA). Samples were mounted using ProLong^® Gold Antifade Mountant with DAPI (Invitrogen, Life Technologies, Carlsbad, CA, USA), and fluorescence visualization was performed by ICTS “NANBIOSIS”, more specifically by the Confocal Microscopy Service (Ciber in Bioengineering, Biomaterials & Nanomedicine (CIBER-BNN)) at the Alcalá University.

Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology. Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit (1:200 for immunoprecipitation; A7470) was purchased from Sigma. Rabbit anti-HA-tag (1:1,000 for immunoblotting; #3724), anti-phospho-JNK-T183/Y185 (1:1,000 for immunoblotting; #4668) and mouse anti-Myc-tag (1:1,000 for immunoblotting; #2276) antibodies were purchased from Cell Signaling Technology. Etoposide (E1383) was purchased from Sigma.

Total proteins were denatured at 100°C by using a Thermocell cooling & heating block (Hangzhou Bioer Technology, China). Equal amounts of denatured proteins (100 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% (weight: volume) skim milk for 1 h at room temperature on a rocking platform shaker, the membrane was incubated in the diluted primary antibody overnight at 4°C. Then, the membrane was incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Finally, the immunoreactive bands on the membrane were detected by using the Chemidoc EQ system (BioRad, USA). Primary antibodies as follows: anti-STAT5a (1:200, Santa Cruz, USA), anti-p-STAT5a (1:200, Santa Cruz, USA), anti-FAK (1:200, Santa Cruz, USA), anti-p-FAK (1:200, Santa Cruz, USA), anti-GSK-3β (1:200, Boster, China), anti-p-GSK-3β (1:200, Santa Cruz, USA), anti-p38α (1:200, Santa Cruz, USA), anti-p-p38α (1:200, Santa Cruz, USA), anti-JNK1 (1:200, Santa Cruz, USA), anti-p-JNK1 (1:200, Boster, China), anti-Src (1:200, Boster, China), anti-p-Src (1:200, Santa Cruz, USA), anti-Caspase-3 (1:200, Santa Cruz, USA), anti-BAX (1:200, Boster, China), anti-Bcl-2 (1:200, Boster, China), anti-p53 (1:200, Boster, China), anti-β-actin (1:2000, Santa Cruz, USA).

PP1, AG1296, Tanshinone IIA, SP600125, and SB202190 were from Sigma (St. Louis, MO, USA). Anti-VCAM-1, anti-β-actin, anti-c-Src, anti-PDGFR, anti-p42, anti-p38, anti-JNK1, anti-c-Jun, and anti-ATF2 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA). Resveratrol was obtained from Sigma (St. Louis, MO, USA). Luciferase assay kit was obtained from Promega (Madison, WI, USA). BCECF/AM was obtained from Molecular Probes (Eugene, OR, USA). Enzymes and other chemicals were obtained from Sigma (St. Louis, MO, USA).