Anti phospho jak2

Proteins were extracted using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS and 50 mM Tris pH. 7,5) with 1 mM PMSF, 10 mM NaF, 2 mM NaVO₃ and Protease Inhibitor cocktail (cat.no. 04-693-1160-001, Roche, USA). For analysis of phosphorylated histones, extracts were prepared in boiling 2% SDS buffer (in 150 mM Tris pH 6,8). Western Blot (WB) analysis was performed as previously described in [17 (link)]. The following antibodies were used: mouse monoclonal antibodies (Mab) to p140Cap already characterized in [17 (link)] (1:500), anti phospho-p130Cas (Tyr410; #4011, 1:1000), anti phospho-Src (Tyr416; #2101, 1:1000), anti phospho-JAK2 (Tyr1007/1008; #3776S, 1:1000), anti γH2AX (Ser139; #2577, 1:1000) and anti H2AX (#2595, 1:1000) from Cell Signaling, Beverly, MA; anti GAPDH (MAB374, 1:8000) from Millipore, Billerica, MA, USA; anti p130Cas (cat.no 610272, 1:2500) from BD Transduction Laboratories, Franklin Lakes, NY; anti Src (B-12, 1:1000) and anti JAK2 (sc-278, 1:1000) from Santa Cruz Biotechnologies, Palo Alto, CA, USA; anti Tubulin (T5168, 1:8000) from Sigma-Aldrich Co, Italy. Secondary antibodies conjugated with peroxidase and nitrocellulose membranes were purchased from GE Healthcare (Buckinghamshire, UK). When appropriate, the membranes were stripped according to manufacturers’ recommendations and re-probed.

U266 cells were stimulated with IL-6 (10 ng/mL) for 20 min in the presence or absence of compounds. Western blot analysis was performed to evaluate STAT3 and JAK2 protein expression in the U266 cell line, as described in previous studies [33 (link)]. The phosphorylation status of STAT3 and JAK2 was examined using anti-phospho-Stat3 (1:1000), anti-Stat3 (1:1000), anti-phospho-Jak2 (1:1000), and anti-Jak2 (1:1000) antibodies (Cell Signaling, Beverly, MA, USA) and then were incubated with the appropriate horseradish peroxide-conjugated secondary antibody (1:5000) at RT. The optical densities of antibody-specific bands were quantified using ImageJ software.

Quercetin, AG490, IL‐6 and DMSO were obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS), trypsin, and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA). Anti‐N‐cadherin, anti‐Vimentin, anti‐E‐cadherin, anti‐MMP‐9, and anti‐Cyclin B1 antibodies were purchased from Abcam (Cambridge, UK). Anti‐Bax, anti‐LC3, anti‐Beclin1, anti‐PCNA and anti‐STAT3, anti‐phospho‐STAT3, anti‐total‐JAK2, anti‐phospho‐JAK2 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Antibody to anti‐P62 was obtained from Proteintech (Chicago, IL).

CCL19 and CCR7 specific monoclonal antibody (mouse antihuman CCR7 antibody) were purchased from R&D System (Minneapolis, MN, USA), PP2 (Src inhibitor) was purchase from Santa Cruz Biotechnology (Dallas, TX, USA), LY294002 (PI3K inhibitor), Tyrphostin A9 (pyk2 inhibitor), and AG490 (JAK2 inhibitor) were purchased from Sigma (St. Louis, MO, USA). The anti-phospho-JAK2, anti-JAK2, anti-phospho-STAT3, anti-STAT3, antivimentin, and anti-E-cadherin were purchases from cell signaling technology (Danvers, MA, USA).

Artesunate (ART), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), Tris base, glycine, NaCl, sodium dodecyl sulfate (SDS), RNase A, DPX mountant for histology, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). Iscove Modified Dulbecco Medium (IMDM), RPMI 1640, and fetal bovine serum (FBS) were obtained from Lonza Group Ltd. (Basel, Switzerland). 0.4% Trypan Blue solution, and antibiotic-antimycotic mixture was obtained from Life Technologies (Grand Island, NY). Anti-phospho-p38, anti-p38, anti-phospho-ERK, anti-ERK, anti-phospho-CREB, anti-CREB, anti-phospho-JAK2, anti-JAK2, anti-procaspase-3, and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-phospho-STAT5, anti-STAT5, anti-SOCS-1, SOCS-1 siRNA, anti-bcl-2, anti-bcl-xL, anti-survivin, anti-cyclin D1, anti-IAP-1, anti-IAP-2, anti-PARP, anti-Ki-67, anti-VEGF, anti-β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). TUNEL (terminal transferase mediated dUTP-fluorescein nick end labeling) assay kit was from Roche Diagnostics GmbH (Mannheim, Germany). Whole-cell lysates of tumor tissues were obtained with T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, USA).