Ab86430

Total protein of cells was extracted using high-efficiency RIPA lysis buffer (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in strict accordance with the instructions. The protein concentration was determined using a BCA kit (20201 ES76, Yeasen Company, Shanghai, China). Then, the protein was separated by polyacrylamide gel electrophoresis and then transferred to PVDF membranes by wet transfer method. Next, the membranes were blocked using 5% BSA at room temperature for 1 h and probed overnight at 4 °C with the following diluted primary antibodies: mouse anti-human GAPDH (ab9425, 1:2500, Abcam, Cambridge, UK), JAK2 (ab39636, 1:1000, Abcam, Cambridge, UK), p-JAK2 (ab195055, 1:1000, Abcam, Cambridge, UK), STAT3 (ab31370, 1:500, Abcam, Cambridge, UK), and p-STAT3 (ab86430, 1:500, Abcam, Cambridge, UK). The membranes were washed for three times (5 min/time) with Tris-buffered saline Tween-20 (TBST), and horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1:20,000, Abcam, Cambridge, UK) was added for 1 h of incubation at room temperature. Then, ImageJ 1.48u software (National Institutes of Health) was performed for protein quantification analysis. The ratio of the gray value of the target band to GAPDH was representative of the relative protein expression. The experiment was repeated three times.

Antibodies to IL-6 (AB6672) and IL-6R (AB83053), STAT3-unphosphorylated (AB68153), STAT3 Serine 727 phosphorylation (AB86430), STAT3 Tyrosine 705 phosphorylation (AB76315), JAK2-unphosphorylated (AB98031 and AB108596), JAK2 phosphorylated-Y1007+Y1008 (AB68268), NF-κB-p105/p50 (AB32360), AKT1 (AB32505), and GAPDH (AB9485) were obtained from Abcam (Cambridge, MA). All HRP-conjugated secondary antibodies were from Invitrogen.