Stat5

The normalized supernatants (5 μg/μL) of colonic tissues were prepared as described in Enzyme-linked Immunosorbent Assay. An equivalent amount of protein in each sample was fractionated onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF) with a Bio-Rad Western blot apparatus. The PVDF membranes were blocked with 5% fat-free milk or 5% bovine serum albumin and then incubated overnight with the following primary antibodies at 4°C. The primary antibodies were JAK1 (1:2000), STAT5 (1:1000), p-STAT5 (1:800), PIAS1 (1:1000), and GAPDH (1:5000) (Abcam, MA, USA). These membranes were treated with the corresponding secondary antibody [HRP-conjugated AffiniPure Goat Anti-Rabbit IgG or HRP-conjugated AffiniPure Goat Anti-Mouse IgG (1:5000–1:10000) (Proteintech, IL, United States] for 1-2 h at room temperature. Subsequently, these membranes were visualized with ECL western blot substrate. The specific protein bands were scanned with a UVP Chen Studio (Analytik Jena, Germany) and quantified using Image-Pro Plus 6.0 software (Media Cybernetic, MD, United States).

Cells were collected and dissolved in RIPA buffer (1% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 10 mM Tris–HCl pH 7.2), Total proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes by electroblotting for 1 h (110 V). The PVDF membranes (Millipore, MA, USA) were blocked in Tris- buffered saline containing 5% nonfat milk and incubated with primary antibody in Tris-buffered saline and Tween (TBST) with 5% nonfat milk over-night at 4 °C. Primary antibodies against the following proteins were used: Erk1/2, p-Erk1/2, Akt, p-Akt, Stat5, p-Stat5, Aven and GAPDH (Abcam, Cambridge, MA, USA); Foxo1, p-Foxo1 (Cell Signaling Technology, Danvers, MA,USA), CD127 (Biolegend) and Bcl2 (PeproTech, Rocky Hill, NJ,USA). The membranes were washed three times for 5 min with TBST and were then incubated with the appropriate secondary antibody in TBST with 5% nonfat milk for 1 h at room temperature. Protein binding was visualized using an enhanced chemiluminescence kit (Pierce) and X-ray films.

Total protein lysates were isolated from the indicated cells using RIPA lysis buffer (JRDUN, Shanghai, China) with an EDTA-free protease inhibitor cocktail (Roche, Germany). An Enhanced BCA protein assay kit (Thermo Fisher, USA) was utilised to measure the protein concentration. A total of 25 μg protein was separated using a 10% SDS-PAGE gel and was transferred to a nitrocellulose membrane (Millipore, USA) overnight. The membranes were probed at 4 °C overnight with the primary antibodies: FOXP3 (1:1000), STAT5 (1:500), p-STAT5 (1:500) (Abcam, UK), and GAPDH (1:2000) (CST, USA) followed by incubation with the secondary antibody (antiserum-HRP tagged goat IgG anti-rabbit 1:1000; Beyotime, China) for 1 h at 37 °C. An enhanced chemiluminescence system (Tanon, China) was used to quantify the protein content. Each sample was tested in triplicate and GAPDH was used as the internal loading control.

Antibodies used for Western blotting including the primary antibodies (JAK3, pSTAT5, STAT5, and GAPDH) and the HRP-conjugated secondary antibody were all procured from Abcam (Cambridge, UK). The procedures were performed as follows: proteins from T cells were initially lysed with RIPA buffer. We then made detection for protein concentrations using a BCA Protein Assay Kit (Thermo Fisher Scientific). Afterwards, 10% SDS-PAGE was used to separate the proteins, followed by transfer into PVDF membranes, in which incubation is with the relevant primary antibodies (1 : 1,000) at 4°C for overnight and then the secondary antibody (1 : 5,000) for 1 h at room temperature. GAPDH was used as the internal control. Immunoblottings were visualized using an ECL detection kit (Amersham Biosciences, Sweden).

Western blotting was performed as previously described [28 (link)]. The primary antibodies were purchased from Santa Cruz Biotechnology [JAK1 (sc-1677)], Cell signaling Technology [JAK2 (#3230), phosho-JAK1 (Tyr1022/1023, #3331), phosho-JAK2 (Tyr1007/1008, #3771), STAT3(#9139), phosho-STAT3 (Tyr705,#9145), phosho-STAT3 (Ser705, #9134), STAT5 (#94205), phosho-STAT5 (Tyr694, #9359)], Abcam [α-SMA (1A4, ab7817)], Bimake [PDGFRβ (A5541), PAI-1 (A5396)], Bioworld [β-actin (#64132)]. β-actin was used as a loading control for all blots.

Ice-cold RIPA buffer (15 mM NaCl, 50 mM Tris HCL PH 7.4, 1 mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate) in the presence of proteases (Roche) and phosphatases (Sigma) inhibitors cocktails was used to cell lysis. After 30 min of ice incubation, the lysates were cleared via centrifugation at 16,000 g for 15 min at 4 °C, and the protein concentration was determined using the BioRad Dc protein assay as recommended by the manufacturer (Bio-Rad, Marnes-La-Coquette, France). Proteins were mixed with 5X Laemmli loading buffer and incubated 10 min at 95 °C. Fifty μg of proteins were migrated in a 10% SDS-PAGE and transferred in a nitrocellulose membrane. The membranes were probed overnight at 4 °C with primary specific antibodies (STAT5, pSTAT5 (Cell Signaling, Danvers, MA, USA), HSC70 (Santa Cruz, Santa Cruz, CA, USA) and FATP2 (Abcam, Cambridge, UK)) after 45 min of non-specific binding sites blocking with TBS 0.1% Tween 5% nonfat milk (pSTAT5) or bovine serum albumin (STAT5, FATP2 and HSC70). The membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories). Immunoblot was revealed using Clarity Western ECL Substrate kit and ChemiDoc imaging system (Bio-Rad).

Equal amounts (10-30 μg) of protein extracts from ischemic quadriceps of the animals (n = 6 for each group) were loaded and separated by SDS-PAGE using 7 or 12 % acrylamide gradients. Proteins were then transferred to nitrocellulose membranes. The membranes were incubated with monoclonal antibodies against JAK2 (1:1000, Abcam), phosphorylated JAK2 (1:1000, Abcam), STAT1 (1: 1000, Cell Signaling), phosphorylated STAT1 (1: 1000, Cell Signaling), STAT3 (1:500, Cell Signaling), phosphorylated STAT3 (1:1000, Cell Signaling), STAT5 (1:1000, Abcam), phosphorylated STAT5 (1:500, Abcam), Akt (1:1000, Cell Signaling), phosphorylated Akt (1:2000, Cell Signaling), JNK (1:500, Sigma), phosphorylated JNK (1:1000, Abcam), ERK1/2 (1:1000, Cell Signaling), and phosphorylated ERK1/2 (1:2000). Signals were detected with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP).