Rabbit anti erk1 2

PDLSCs were washed with ice‐cold PBS and lysed using a RIPA lysis buffer containing 1% PMSF (Solarbio) and 1% phosphatase inhibitor (Boster, Wuhan, China) and then were centrifuged at 12,000 g for 10 min. Protein concentration was measured by a BCA Protein Assay Kit. Proteins were added with SDS‐PAGE loading buffer and then denaturated at 100°C for 5 min. 20 μg/lane proteins were loaded to SDS‐PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with non‐fat dry milk for 1 hr, blotted primary antibodies overnight at 4°C and subsequently incubated with horseradish peroxidase‐labelled secondary antibodies (1:2000; Beyotime, Shanghai, China) for 1 hr at room temperature. The membrane was washed three times in tris‐buffered saline with 1‰ Tween20 (TBST). The immunoreactive bands were visualized using enhanced chemiluminescence reagents (Millipore). The level of each protein was normalized to GAPDH before statistical analysis. Image J 1.44 software (NIH, Bethesda, Maryland, USA) was used to quantify the protein expression. The primary antibodies used were as follows: rabbit anti‐phospho‐ERK1/2 (1:1000; Cell Signaling Technology, Danvers MA, USA), rabbit anti‐ERK1/2 (1:2000; Cell Signaling Technology), rabbit anti‐phospho‐p38 (1:1000; Cell Signaling Technology) and GAPDH (1:10,000; Proteintech, Chicago, IN, USA).

Western blot analysis was performed as previously described, with slight modifications [11 (link)]. Briefly, HUVEC lysates were lysed using RIPA buffer supplemented with protease inhibitor (Cell Signaling 5871S). Proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel, transferred to a PVDF membrane (GE Healthcare, RPN303F), and blocked in 5% bovine serum albumin (BSA) in PBS with 1% tween-20 (Sigma P2287) for 1h at RT. Primary antibodies used were: rabbit anti-phospho-Smad1/5 (1:1000, Cell Signaling 9516), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1000, Cell Signaling 4370), rabbit anti-ERK 1/2 (1:1000, Cell Signaling 4695), mouse anti-HIF1α (1:500, Novus biologicals NB100-105), mouse anti-p53 (1:1000, Abcam ab1101) and rabbit anti-p53 (1:500, Abcam ab131442). Membranes were incubated with primary antibodies diluted in 1% BSA overnight at 4°C. Signal was detected with horseradish peroxidase (HRP) anti-rabbit (1:5000, Invitrogen G-21234) or HRP anti-mouse (1:30,000, Invitrogen 81–6720), and imaged via Clarity Western ECL Substrate (Bio-Rad 170–5061). Full original blots are shown (S6 Fig).

Cellular lysates and western blotting assays were performed as previously depicted [20 (link)–22 (link)]. The following antibodies were used: mouse anti-β-actin (Sigma, St. Louis, MO, USA), rabbit anti-Mxi1-0 (Invitrogen, Carlsbad, CA), rabbit anti-ERK1/2, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti- phospho-Cdk1(Tyr15) and anti-phospho-Chk1(Ser345) (Cell Signaling Technology, Boston, MA, USA), rabbit anti-CyclinB1(Proteintech,Wuhan, China). Antiserum against Mxi1 and Mxi1-0 were raised in rabbits by injection of Keyhole Limpet Hemocyanin (KLH)–conjugated, synthetic peptides (CEAAEFLERRE for Mxi1, GKRGRPRKEARCE for Mxi1-0), corresponding to amino acids 13–23 of Mxi1 and amino acids 2–14 of Mxi1-0, respectively. Antibodies were affinity-purified by Invitrogen (Carlsbad, CA, USA).

The immunoblotting analyses were conducted as described previously^{40 (link)}. In brief, each specimen was electrophoresed using 12% SDS-polyacrylamide gels. The proteins on the SDS gels were then electrophoretically transferred to nitrocellulose membranes. Following the blocking, the membranes were incubated at 4 °C overnight under gentle agitation with rabbit anti-Akt antibody and rabbit anti-pAkt (Ser 473) antibody (1:1000; Cell Signaling, Danvers, MA, USA), rabbit anti-ERK1/2 (1:1000; Cell Signaling Technology) and rabbit anti-pERK1/2 (1:1000; Cell Signaling Technology). After being washed, the membranes were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit (1:1000; GE Healthcare, Tokyo, Japan) for 2 h at room temperature. Subsequently, the membrane-bound, HRP-labeled antibodies were detected using an enhanced chemiluminescence detection system (ECK lit, GE Healthcare, Tokyo, Japan) with an image analyzer (LAS-1000; Fuji Photo Film, Tokyo, Japan).