β actin

Western blotting was performed as previously described [27 (link)]. Ca9-22 cells were lysed in CelLytic M lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor cocktail (Nacalai Tesque). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories), and probed with rabbit anti-human ANGPTL2 polyclonal antibody (Proteintech Group, Chicago, IL, USA, Cat:12316-1-AP) or rabbit anti-human β-actin monoclonal antibody (Cell Signaling Technologies, Beverly, MA, USA, Cat:#7074S). β-actin (Cell Signaling Technologies, Cat:#3700S) was the loading control. Goat anti-rabbit IgG (Cell Signaling Technologies, Cat:#7076S) and goat anti-mouse IgG were used to detect ANGPTL2 and β-actin, respectively.

PTECs were lysed in RIPA buffer [50 mM Tris·HCl, pH 7.3; 150 mM NaCl; 0.1 mM EDTA; 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; 0.2% NaF; and 100 mM Na3VO4] supplemented with complete protease inhibitors (Roche Applied Science, Indianapolis, IN). The kidney homogenate was centrifuged at 12,000 × g for 30 minutes at 4 °C, and the protein concentration of the supernatant was determined by the Bradford method. Different amounts of extracted protein were separated by SDS-PAGE and transferred onto Immobilon-FL 0.4-μm polyvinylidene difluoride membranes (Millipore). Tris-buffered saline containing 0.1% Tween 20 was used as the washing buffer. After probing with primary antibodies, anti-rabbit (1:5,000; Cell Signaling Technology, Danvers, MA) and anti-mouse (1:6,000 for β-actin; Cell Signaling Technology) antibodies were used as secondary antibodies. Detection of labeled proteins was performed with an enhanced chemiluminescence system (ECLTM PRN 2106; Amersham Pharmacia Biotech, Buckinghamshire, UK). The band intensities were analyzed using a gel documentation system (Bio-Rad Gel Doc 1000 and Multi-Analyst version 1.1)^{17 (link)}.
Western immunoblotting was performed using primary antibodies against collagen 1 (Abcam), fibronectin (Santa Cruz Biotechnology, Dallas, TX), αSMA (Abcam), and β-actin (Cell Signaling Technology).

Protein was extracted from intestinal mucosal samples and IEC‐6 cells. Cytoplasmic protein extraction was performed with a commercially available kit (Beyotime, Beijing, China). For RIP1 immunoprecipitation, 2 mg protein were pre‐cleared with protein G‐Sepharose beads for 2 hrs and incubated with the RIP1 antibody or control IgG O/N at 4°C. For western blot, total protein was electrophoresed on 10% or 12% SDS‐PAGE and transferred to PVDF membrane. After blocking, the blots were further incubated overnight 4°C with cleaved Caspase‐3 (9661; Cell Signaling Technology), RIP1 (610459; BD Biosciences, Franklin Lakes, NJ, USA), RIP3 (ADI‐905‐242; Enzo Lifescience), MLKL (sc‐165025; Santa Cruz, Dallas, TX, USA), HMGB1 (ab18256; Abcam), Toll‐like receptor 4 (TLR4, sc‐10741; Santa Cruz), Receptor for advanced glycation end products (RAGE, sc‐5563; Santa Cruz). The membranes were washed and incubated with the secondary antibodies (IRDye 800CW Goat antimouse/Rabbit IgG, Licor‐biosciences, Lincoln, NE, USA). After additional washing, the blots were analysed by the LICOR Odyssey infrared imaging system. β‐actin (4970; Cell Signaling Technology) served as the internal control, and the results were expressed as a ratio relative to β‐actin.

To analyse the mRNA expression of angiogenic relative genes in transplanted human ADSCs, total RNA of tissue was extracted and the expression of angiogenesis related human genes was detected by quantitative RT‐PCR as described previously in 2.3. To measure the proteins extents, the tissues were lysed in Laemmli Sample Buffer (Bio‐Rad) and the total proteins obtained. The proteins extents of HIF‐1α, VEGF, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Collagen III, Elastin and β‐Actin (Cell Signaling Technology) were detected as described previously in 2.4, and the band intensity was normalized with β‐Actin as the endogenous control.

Cell monolayers were washed once with DPBS, then lysed on ice for 15 min in 100–200 μL RIPA cell lysis buffer (Sigma) supplemented with protease and phosphatase inhibitors (Roche, Brighton, MA, USA). Protein was determined by the BCA (Thermo Scientific, Waltham, MA, USA) assay and equal amounts of proteins were resolved by SDS denaturing polyacrylamide electrophoresis on 3–8% Tris-Acetate (large proteins), 4–12% Bis-Tris or 10–20% Tris-Glycine Novex gels (small proteins) (Invitrogen, Waltham, MA, USA), transferred to nitrocellulose via iBLOT, blocked with Tris HCL buffered saline (TBS) pH 7.4 LiCor Blocker, and incubated overnight at 4 °C, with primary antibody diluted in Blocker, 0.1% Tween 20. The blots were washed with TBS, 0.1% Tween 20, incubated for 1 h at RT with secondary antibody LiCor that was 800W-labeled and diluted at a ratio of 1:15,000 in Blocker, 0.2% Tween 20. It was then re-washed and dried and fluorescent image was acquired using an Odyssey IR imaging system (LiCor Biosciences Lincoln, NE, USA). Antibodies used for immunoblots, β-Actin, pS6 (Ser235/236), S6, TSC2, mTOR, pmTOR (Ser2448), p4E-BP1 (Thr37/46), 4EBP1, Akt, pAkt (Ser273), and β-Actin were all from Cell Signaling. Antibody to peIF4E (Ser209) (76256) was from ABCAm (Woburn, MA, USA). All primary antibodies were used at 1:1,000 dilutions except β-Actin, which was at 1:15,000 dilution.