Anti p jak2

Tissue samples and HCC cells were lysed using RIPA lysis buffer supplemented with PMSF (1:100, G-CLONE) to obtain total proteins. The proteins concentration was estimated using the BCA protein quantification kit (Solarbio). Equal amounts of protein were subjected to SDS-PAGE, proteins were then transferred onto PVDF membranes (PerkinElmer). After blocking with skimmed milk or BSA, the membranes were incubated with the primary antibodies overnight at 4 ℃. Primary antibodies were as follows: anti-EVA1A (1:500, Abcam), anti-TP53 (1:1000, OriGene), anti-BAX(1:2000, OriGene), anti-BCL-2 (1:2000, Bioss), anti-p-JAK2 (1:1000, Cell Signaling Technology), anti-p-STAT3 (1:1000, Cell Signaling Technology), anti-MMP-9 (1:1000, Cell Signaling Technology), anti-LC3 (1:1000, MBL), anti-p62 (1:1000, Proteintech), anti-Beclin1 (1:2000, Proteintech) and anti-β-actin (1:1000, Servicebio). And then the blot was incubated with peroxidase-conjugated sheep anti-rabbit IgG (1:3000, Servicebio). Protein bands were detected using the ECL system. Each independent experiment was performed at least three times.

House dust mites (HDM, ALK-Abello A/S, Denmar), Recombinant Human long-isoform TSLP (lTSLP) was obtained from R&D systems. Synthetic sfTSLP peptides (63aa: MFAMKTKAALAI WCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ) were prepared by China Peptides (Shanghai, China). 3-PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) and BAY 87-2243 (Selleck, China). Rabbit anti-HIF-1α, anti-LDHA, anti-PHD and anti-VHL (proteintech, China), Rabbit anti-STAT5, anti-p-STAT5, anti-JAK1, anti-p-JAK1, anti-JAK2and anti-p-JAK2 (Cell Signaling Technology, USA), Rabbit anti-TSLPR and mouse anti-IL-7R (santa curz, USA). Rabbit anti-TSLP (Abcam, USA).

Tumor homogenate or cell lysate was prepared with RIPA buffer supplemented with complete protease inhibitors. Equal amounts of proteins were separated in 8% or 12% SDS-PAGE and immunoblotted with the following primary antibodies: anti-arginase-1 (MABS388, Merck-Millipore, Billerica, MA, USA), anti-CCR-2 (ab125686, Abcam), anti-PCNA (NA03, Merck-Millipore), anti-p-JAK2 (4406T, Cell Signaling Technology, Beverly, MA, USA), anti-JAK2 (3230T, Cell Signaling Technology), anti-p-STAT3 (9145T, Cell Signaling Technology), anti-STAT3 (4904T, Cell Signaling Technology), anti-fibronectin (ab32419, Abcam), anti-vimentin (#5741P, Cell Signaling Technology), anti-snail2 (#3879P, Cell Signaling Technology), and anti-β-actin (ab8226, Abcam), followed by the secondary HRP-conjugated antibodies.

Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton-X100 supplemented with 1 mM phenylmethylsulfonyl fluoride before use). Cell lysate in SDS loading buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.1% bromophenol blue) was boiled and analyzed by SDS-PAGE (Bio-Rad) and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes with transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, and 80% ddH₂O, pH 8.3). Then the PDVF membranes were blocked with 2% milk (2% bovine serum albumin for phosphorylated antibody) with TBST buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.6, and 0.1% Tween-20) for 1h before incubation with primary antibodies for 2 h at room temperature or 4 °C overnight. After washing with TBST, membranes were incubated with secondary antibody for 1 h and then washed three times with TBST. Cells need to be treated with 50 ng/mL IFN-γ 48 h in advance for phosphorylated protein detection. The antibodies, including anti-Jak2 (Cell Signaling Technology Cat# 3230, RRID: AB_2128522), anti-pJak2 (Cell Signaling Technology Cat# 3771, RRID: AB_330403), anti-STAT3 (Cell Signaling Technology Cat# 12640, RRID: AB_2629499), anti-pSTAT3 (Fluidigm Cat# 3158005, RRID: AB_2661827), anti-TPM4(Abcam Cat# 181085), anti-SOX2 (Abcam Cat# ab92494, RRID: AB_10585428), anti-GAPDH (BioXcell, #BX-008) were used in this study.

Cytosolic proteins were extracted using RIPA buffer containing Halt protease/phosphatase inhibitor cocktail (Thermo). Membrane fraction of cell lysates were extracted using Mem-PER Plus membrane protein extraction kit (Thermo). For immunoblotting, proteins (15–30 μg for each sample) were separated by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes (Biorad) and probed with the following antibodies: anti-pSTAT1_Y701, anti-STAT1, anti-pSTAT3_Y705, anti-pSTAT3_S727, anti-STAT3, anti-p-JAK2, anti-JAK2 (Cell signaling technology), anti-nephrin (Progen), anti-podocin, anti-Na/K ATPase (Abcam) and anti-β-actin (Sigma). Subsequently, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Thermo). Reactive proteins on membranes were visualized using a SuperSignal West Pico Chemiluminescent substrate (Thermo). These membranes were then exposed to an Amersham Imager 600 (GE Healthcare).

Western blotting analysis was performed as described previously [15 (link)]. Briefly, cells treated with CT for 48 h were lysed in RIPA buffer (Beyotime, Beijing, China). The protein concentrations were quantitated with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Frederick, MD, USA). Total protein 30 µg/lane were loaded onto SDS–polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ). The membranes were blocked and incubated with (1:1000) rabbit anti-p-STAT3 (Cell Signaling, Cat: 9145L, Tyr 705), anti-STAT3 (Cell Signaling, Cat: 4904S), anti-p-JAK2 (Cell Signaling, Cat: 8082), anti-JAK2 (Cell Signaling, Cat: 3230), anti-I-kBα (Cell Signaling, Cat: 9242), anti-GAPDH (Cell Signaling, Cat: 2118) overnight at 4 °C and (1:2000) horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Cat: 70,741) for 1 h at room temperature. The protein bands were visualized using the G-BOX Chemi system (Syngene, Frederick, MD).

Western blotting was performed to analyze the protein level of FAM134B, p-JAK2 and p-STAT3 in our study. Anti-FAM134B antibody (Cell Signaling, Danvers, MA, USA, 1:500), anti-p-JAK2(1:500), anti-JAK2(1:500), anti-p-STAT3(1:1000), anti-STAT3(1:500) (Cell Signaling, Danvers, MA, USA). The membranes were then stripped and re-probed with an anti- β-actin monoclonal antibody (Cell Signaling, 1:3000) as a loading control.