Ripa lysis buffer p0013b

Bovine serum albumin (BSA, A4161), 5-hydroxytryptamine (H9523), and eletriptan hydrobromide (1234453) were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Fetal bovine serum (FBS, 10099-141) and α-minimum essential medium (α-MEM, 12561056) were obtained from Gibco Co. Ltd. (Waltham, MA, USA). RIPA lysis buffer (P0013B) was purchased from Beyotime Co. Ltd. (Haimen, China). Ampicillin (A100741), vancomycin (A100990), neomycin (A610366), metronidazole (A600633), and amphotericin B (A610030) were purchased from Sangon Biotech Co. Ltd. (Shanghai, China).

This study acquired 20 (R)-G-Rg3 (purity≥98.0%, HPLC) and evaluated the quality according to our prior description. In addition, the present work obtained D-gal from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), whereas BCA protein assay kit (P0010) and RIPA lysis buffer (P0013B) in Beyotime Biotechnology Co., Ltd. (Jiangsu, China). Additionally, we purchased alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), high/low density lipoprotein cholesterol (LDL-H/LDL-C), and hematoxylin-eosin (H&E) kits in Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). The present work acquired Mouse IL-1β, Mouse IL-6, Mouse IL-18, Mouse TNF-α ELISA kits in Multi Science Biotechnology Co., Ltd. (Hangzhou, China).

Western blotting was performed as described previously [13 (link)]. Briefly, calluses were ground in liquid nitrogen and incubated with RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology, Shanghai) at 4 °C for 30 min, with shocking every 5 min. Samples were then centrifuged for 10 min at 14,000 rpm at 4 °C. Denatured proteins were separated by SDS-PAGE and transferred onto a PVDF membrane, which was blocked with 5% skim milk in 1× Tris-buffered saline Tween-20 (TBST, Applygen Technologies, Beijing). The membrane was then incubated with a primary antibody (anti-BMP2, anti-TGF-β1, anti-VEGF, or anti-β-actin (all from Abcam, MA, USA)) followed by a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Beyotime Institute of Biotechnology, Shanghai). The membrane was then exposed to Kodak Biomax-Ml film (Eastman Kodak Co., USA). Signals were quantified by densitometry.

Total proteins were extracted using radio‐immunoprecipitation assay (RIPA) lysis buffer (P0013B; Beyotime Biotechnology Co., Ltd.) containing phenylmethylsulfonyl and phosphatase inhibitors. After a 30‐min incubation on ice, the samples were centrifuged at 12 000 rpm for 10 minutes at 4°C, followed by the supernatant collection. A bicinchoninic acid (BCA) kit (Beyotime Biotechnology Co., Ltd.) was utilized to determine protein concentration. Next, 30 µg protein was separated by sodium dodecyl sulfonate‐polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane using wet transfer apparatus. After that, the membrane was blocked with 5% skimmed milk in Tris‐buffered saline Tween (TBST) buffer for 1.5 hours and incubated with the primary antibody to MRP1 (1:500; ab32574, Abcam) at 4°C overnight. On the following day, the membrane was further incubated with the secondary antibody, horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit antibody to immunoglobulin G (IgG; 1:2000‐50000; ab205718) for 2 hours at room temperature. Subsequently, the protein bands were visualized with an enhanced chemiluminescence (ECL) reagent, which were imaged on SmartView Pro 2000 (UVCI‐2100; Major Science). Image analysis was followed using Quantity One software. The target proteins were quantified as relative gray values against the internal reference GAPDH.

Western blot was adopted to evaluate the expression level of AQP4 in ipsilateral hippocampus. Whole cells of samples were lysed with RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology, Jiangsu, China) and the protein was determined with a BCA kit (Beyotime Institute of Biotechnology, Jiangsu, China). Equal amount of protein was separated by 10% Tris-glycine SDS-PAGE polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Invitrogen, USA). After blocking for 1.5 h with a 5% solution of skim milk (232100, BD-Difco, USA), the membranes were incubated with the primary antibodies polyclonal rabbit anti-AQP4 (1 : 1000; ab46182, Abcam, USA) or polyclonal rabbit anti-GAPDH (1 : 1000; AP0063, Bioworld Technology, USA) at 4°C overnight, followed by the horseradish peroxidase- (HRP-) linked anti-goat antibody (1 : 10000; Boyun Biotech, Shanghai, China) for 30–60 min at room temperature. After washing with Tris-buffered saline with 0.1% Tween-20 (Beyotime Institute of Biotechnology, Jiangsu, China), the relative intensity of protein signals was normalized to the corresponding GAPDH intensity and was quantified by densitometric analysis with the use of Quantity One software (Bio-Rad Laboratories, USA).

To analyze the effects of IGFBP3 on the promigratory signaling pathway, hBMSCs were treated with culture medium containing 25 ng/ml IGFBP3 with or without pretreatment with 2 µM of SB505124 for inhibition of TβR I/II or p‐Smad 2/3, 500 nM BMS CCR2 22 for inhibition of CCR2. The hBMSCs were collected with or without IGFBP3 treatment for 6 hr, 12 hr, 1, 2, and 3 days, respectively. Total protein was extracted with 100 µl RIPA lysis buffer (P0013B, Beyotime, Jiangsu, China), subjected to SDS–PAGE, transferred on nitrocellulose membranes (Millipore, Billerica) and probed with specifics primary antibodies against p‐Smad 2/3 (Cell Signaling Technology), Smad 2/3(Santa Cruz Biotechnology, Texas), CCR2 (Santa Cruz Biotechnology) or TβR I/II (Santa Cruz Biotechnology), or GAPDH (Beyotime, Jiangsu, China) at 1: 500 dilution overnight at 4°C. Immunoreactive protein bands were visualized using ECL chemiluminescence detection plus a Western blot detection system (Bio‐Rad). The intensity ratio was the relative expression of p‐Smad2/3, Smad 2/3, TβR I, TβR II, and CCR2 normalized to GAPDH.