P jak2

We obtained CB2 antibody (1:500) from Abcam Inc. (Cambridge, MA). We purchased CB1 (1:500), total STAT1 (1:1000), total STAT3 (1:1000), iNOS, NOX3, and TRPV1 (1:300) from Santa Cruz Biotechnology (Santa Cruz, CA). We bought p-STAT1^{Ser 727} (1:500), p-STAT3^{Tyr 705} (1:500), p-JAK2 (1:1000), from Cell Signaling Technology Inc. (Danvers, MA). We purchased secondary antibodies; goat anti-rabbit, donkey anti-goat and goat anti-mouse from Life Technology (Eugene, OR), and fluorescent tagged (dylight 488 and TRITC) secondary antibodies (1:500) from Jackson Immuno Laboratories (West Grove, PA).

Cells after SC99 treatment were prepared for immunoblotting according to our previous method [35 (link), 40 (link)]. A specific primary antibody against cyclin D₂ (CCND2) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antibodies against Bcl-2, Bcl-xL, Caspase-3, STAT3, p-STAT3(Tyr705), c-Src, p-Src, JAK2, p-JAK2, E2F-1, AKT, p-AKT, ERK, p-ERK, mTOR, p-mTOR, and PARP were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against VEGF, β-actin, α-tubulin, anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems (Minneapolis, MN). Anti-Myc antibody and GAPDH was purchased from Sigma (St. Louis, MO).

After 72 h of transfection, the cells were collected into 1.5MLEP tubes. The cells were lysed with protein lysates consisting of RIPA: PMSF = 100 : 1. The protein concentration was determined using the BCA protein assay kit. After adding the protein sample buffer, the cells were boiled in a 100°C- water bath for 10 min to denaturate the proteins.
Start electrophoresis after adding protein and maker to the sample tank. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Solarbio, Beijing, China). PVDF membrane was cut according to the molecular weight of the required proteins. And then the PVDF membrane was sealed in 5% skimmed milk powder for 1 h and washed with TBST for 3 times. The antibody (GAPDH, OSMR, JAK2, STAT3, pJAK2, pSTAT3) (Cell Signaling Technology, USA) was incubated and placed in a shaker for 4°C overnight, and the anti-rabbit IgG sheep antibody (Cell Signaling Technology, USA) incubated for exposure the next day. The chromogenic condition of the protein was observed and photographed.

Cells were lyzed with RIPA buffer and total 30 μg of protein was loaded on 6–12% SDS-PAGE. After transferring to PVDF membranes, each membrane was blotted with the appropriate antibodies. Anti-PARP, -p-EGFR, -EGFR, -p-STAT3, -STAT3, -p-JAK1, -p-JAK2, -p-AKT, and -AKT antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p-SRC, -SRC, -p-ERK1/2, -ERK1/2, -VEGF, -Cyclin D, -MMP-9, -Survivin, and -Tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immunofluorescence assays for p-STAT3 nuclear translocation in MDA-MB-231 cells were done with anti-p-STAT3 antibody and anti-Alexa Fluor-488 antibody (Invitrogen, Eugene, OR, USA). For the counter staining, TOPRO-3 (Invitrogen, Eugene, OR, USA) was used to stain the nucleus. Images were obtained with Olympus FV10i Self-Contained Confocal Laser System.

Whole-cell Brij protein lysis, nuclear and cytosolic protein extractions, and Western-blot analyses were performed as described [46] (link), [65] (link). Antibodies used for detection of the respective proteins and their relevant dilutions were: pSTAT5 (#9351, Cell Signaling Technology; 1∶1000), STAT5A (L-20, sc-1081, Santa-Cruz Biotechnology; 1∶1000), STAT5B (G-2, sc-1656, Santa-Cruz Biotechnology; 1∶200), STAT5A+B (C-17, sc-835, Santa-Cruz Biotechnology; 1∶1000), pJAK2 (Cell Signaling Technology, #3771; 1∶200), JAK2 (#3230, Cell Signaling Technology; 1∶500), α-tubulin (DM1A, sc-32293, Santa-Cruz Biotechnology; 1∶200), Anti-Rabbit IgG-Peroxidase (SIGMA A0545; 1∶10,000), Anti-Mouse IgG-Peroxidase (SIGMA A8924; 1∶10,000). Apparent molecular weight of detected proteins was as predicted by the antibody manufacturers, i.e. 125 kDa for JAK2, 90 kDa for STAT5 (with STAT5A running slightly slower than STAT5B in SDS-PAGE, as confirmed by the STAT5A+B immunoblots) and 55 kDa for α-tubulin. Chemoluminescence detection was performed using Amersham ECL Prime (RPN2232, GE Healthcare Life Sciences) or SuperSignal West Femto (34095, Thermo Fisher Scientific) for weaker signals, and images were captured on an ImageQuant LAS 4000 mini imaging system (GE Healthcare Life Sciences). Immunoblots shown are representative of at least three independent experiments.