Ab128008

As described previously [48 (link)], proteins (40-50 mg) from cells or tissues were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (PVDF; Bio-Rad, USA). The membranes were blocked and then probed with antibodies against IL-6 (ab6672) or IL-6R (ab128008) (Abcam, Cambridge, MA, USA). p-STAT3 antibody (tyr705) (#4113) was purchased from Cell Signaling Technology, Danvers, MA, USA. The following antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Mcl-1 (sc-819), Survivin (sc-8807) and Bcl-2 (sc-7382). Antibodies against Bax (B3428) and β-actin (A9044) were obtained from Sigma (St. Louis, MO). After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies.

Total proteins were extracted using RIPA buffer (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s protocol, separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (MilliporeSigma, Burlington, MA, USA). The blots were incubated with primary antibodies, followed by the incubation with the appropriate horse radish peroxidase conjugated secondary antibody, detected with Bio-Rad® ChemiDoc® MP(Bio-Rad), and were analyzed using Quantity One 4.6 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primary antibodies used included anti-E-cadherin (cat. no. 1416, Abcam, Cambridge, United Kingdom), anti-N-cadherin (ab18203, Abcam, Cambridge, MA, USA), anti-Vimentin (ab8978, Abcam, Cambridge, MA, USA), anti-GAPDH antibody (ab8245, Abcam, Cambridge, MA, USA), anti-PTEN antibody (ab32199, Abcam Cambridge, MA, USA), anti-IL6R antibody (ab128008, Abcam, Cambridge, MA, USA), anti-Akt antibody (10176-2-AP, proteintech, Wuhan, China), anti-p-Akt antibody (66444-1-Ig, proteintech), anti-ERK antibody (16443-1-AP, proteintech), anti-p-ERK antibody (#3510, Cell Signaling Technology, Danvers, MA, USA) anti-p-STAT3 antibody (#9145, Cell Signaling Technology), anti-p-JAK2 antibody (#3771, Cell Signaling Technology), and anti-β-actin antibody (ab8226, Abcam).

Proteins were extracted by incubation with RIPA buffer and protease inhibitor PMSF. Protein concentration was determined by using BCA assay kit (RTP7102, Real-Times Biotechnology Co., Ltd., China). Then, 20 µg of proteins were separated on 10% SDS-Page and transferred to polyvinylidene difluoride membranes. After blocking with 5% skimmed milk for 1 h, the membranes were probed with the following antibodies: rabbit anti-IL-6R (1:1000, ab128008, Abcam, USA) or rabbit anti-β-actin (1:5000, ab129348, Abcam) at 4°C overnight. For detection, goat anti-rabbit (1:3000, ab6721, Abcam) secondary antibodies conjugated to horseradish fluoride were used. Signal detection was performed using chemiluminescence reaction (ECL; ab65623, Abcam). The acquired images were analyzed by Image Lab 3.0 (Bio-Rad Laboratories, USA) and the relative protein expression is reported as the densitometric value ratio of IL-6R band to β-actin band.

Immunofluorescence staining was carried out as previously described [20 (link), 22 (link)]. Antibodies (1:100) against IL6R (ab128008, Abcam) and NFAT1 (ab2722, Abcam) were used to detect the co-expression of these two proteins.

Proteins were leached by RIPA lysis buffer (Beyotime, Shanghai, China) and quantified by an Enhanced BCA Protein Assay Kit (Beyotime). Total 20 μg protein was split up by SDS-PAGE and transferred to PVDF membranes (Beyotime). Blocked by 5% concentration of bovine serum albumin (BSA, Sigma-Aldrich) at 37 °C for 0.5 h, the membranes were cultivated with primary antibodies at 4 °C overnight (using GAPDH as internal reference). Then, secondary antibody was added and the culture continued at room temperature for another 1 h. Washed three times by TBST, HRP-labeled proteins were introduced by BeyoECL Star Kit (Beyotime) and filmed. The primary antibodies were as follows: rabbit anti-IL6 (ab6672, 1:2000, Abcam, Cambridge, MA, USA), rabbit anti-IL6R (ab128008, 1:500), rabbit anti-pan-Akt (ab8805, 1:500), rabbit anti-pan-Akt (phospho T308) (ab38449, 1:500), and rabbit anti--GAPDH (ab181603, 1:10000). The secondary antibody was HRPlabeled goat anti-rabbit IgG (ab205718, 1:2000).

Western blotting analysis was performed as described previously [20 (link)]. Briefly, a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China) was used to extract total protein. An equivalent amount of protein from each sample was electrophoresed and transferred to a nitrocellulose membrane and blocked with 2% bovine serum albumin (BSA). The membranes were then incubated with anti-IL6 (1:2000, ab6672, Abxam), IL6R (1:1000, ab128008, Abcam) or NFAT1 (1:1000, ab2722, Abcam) at 4°C overnight. Membranes were washed four times with TBST and incubated with the appropriate secondary antibody. Bands were detected using a chemiluminescence kit (Beyotime Biotechnology, Beijing, China) and quantified with Image J (National Institutes of Health, Bethesda, MD, USA).