Rabbit anti mouse igg

Total cell lysates were prepared and separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as previously described (30 (link)). Briefly, intestinal tissues were homogenized in a 1.5-mL tube containing 250 μL of 1× cell lysis buffer (#9803; CST, Danvers, MS, USA) supplemented with a cocktail of complete protease inhibitors (Roche, Basel, Switzerland) using an electric homogenizer for 1 min on ice. After centrifugation, supernatants were mixed with an equal volume of 2× sample buffer [125 mM Tris-HCl, 0.01% bromophenol blue, 4% SDS, 20% glycerol, and 200 mM dithiothreitol (DTT)] and boiled for 10 min. Total protein concentration was measured using the DC™ Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Tissue extracts were separated using SDS-PAGE and immunoblotted with the following antibodies: rabbit anti-SULT2B1b [custom-made (26 (link)); 1:1,000], and goat anti-β-actin (#sc-1616; 1:2,000, Santa Cruz Biotechnology, TX, USA). The following horseradish peroxidase-conjugated secondary antibodies were used: mouse anti-rabbit IgG (#sc-2357; 1:2,000, Santa Cruz Biotechnology), and mouse anti-goat IgG (#sc-2354; 1:2,000, Santa Cruz Biotechnology).

Solute carrier family 27 member 2 (FATP2) and microsomal triglyceride transfer protein (MTTP) were assessed by western blot. The protein concentration was determined according to the Bradford protocol [20 (link)]. Protein samples (40 μg) were denaturalised at 95 °C for 3 min in Laemmli buffer [22 (link)] and loaded into 4–15% Mini-PROTEAN TGX Precast Gels (BioRad, Hercules, CA, USA). The proteins were then transferred onto PVDF membranes (Millipore, MA, USA) by electroblotting and later blocked with 5% casein and 0.5% bovine serum albumin (BSA) PBS-tween buffer for 2 h at room temperature. Subsequently, membranes were incubated with anti-FATP2 (1:500) (Santa Cruz Biotech, Santa Cruz, CA, USA), anti-MTTP (1:500) (Abcam, Cambridge, UK) and anti-α-tubulin (1:2000) (Cell Signaling, Beverly, MA, USA) for at least 1 h at room temperature and kept at 4 °C overnight. After washing, membranes were incubated with secondary antibodies (mouse anti-goat IgG; 1:5000 and mouse anti-rabbit IgG; 1:5000) (Santa Cruz Biotech, Santa Cruz, CA, USA); the immunoreactive proteins were detected by the Forte Western HRP substrate (Millipore; Burlington, MA, USA), and the blots were imaged by scanning with the ChemiDoc™MP Imaging System (Bio-Rad, Hercules, CA, USA). α-tubulin was used as housekeeping.

Sections were deparaffinized in Histoclear and rehydrated through ethanols. Peroxidase blocked (3% hydrogen peroxide in methanol) for 15 min and washed in PBS. Antigen retrieval was achieved at 95 °C for 20 min in citrate buffer, before blocking (10% NCS, 0.1% BSA, 0.4% Triton‐X‐100) for 1 h. Primary antibody (S100, Abcam, ab52642, 1:100) was then applied for 1 hand washed in PBS before a biotin conjugated secondary antibody (Mouse anti‐rabbit IgG, Santa Cruz Biotechnology, California, US, sc2491, 1:200) was added for 1 h. Slides were then washed in PBS and ABC reagent (Santa Cruz Biotechnology) for 30 min before being developed in DAB reagent (Vector Laboratories). Slides were washed in dH₂O and counterstained in Gill's Hematoxylin (Sigma‐Aldrich) before being dehydrated, cleared in Histoclear and mounted using Omnimount.

The testes were dissected out and homogenized in pre-chilled RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% Sodium deoxicholate, 0.1% SDS, 1% Tween-100, 5 mM EDTA, 1 mM EGTA, 1 mM PMSF). The lysate were centrifuged at 12,000 g for 15 min at 4 °C to pellet the debris. Supernatant solutions were collected as whole cytoplasm protein for analyzing PCNA. Protein concentrations were determined by the Coomassie blue protein binding method using bovine serum albumin as standard. Then samples containing equal amounts of protein (50 μg) were boiled in protein loading buffer for 10 min, separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 3% BSA in Tris-buffer saline. Then the membranes were incubated at 4 °C overnight with the PCNA antibody (1:1000) and β-actin antibody (1:1000), respectively (Santa Cruz Biotechnologies, Santa Cruz, CA). After washing, the membranes were incubated with a horseradish peroxidase conjugated secondary antibody (mouse anti-rabbit IgG, 1:5000; rabbit anti-goat IgG, 1:5000) (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h at room temperature and were visualized using an enzyme-linked chemiluminescence reaction by imager instrument (Fujifilm, Las-3000). Relative intensities of the bands were quantified by Image-Pro Express 6.0 software.

Protein samples were separated in polyacrylamide gels (7 to 12%, depending on the target protein) and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Western blotting (WB) was performed as described previously [56 (link)]. Primary antibodies used were anti-CD6 mAb (MEM98, EXBIO), anti-RasGAP mAb (B4F8, Santa Cruz Biotechnology), anti-p21 Ras mAb (Cytoskeleton, Inc), anti-α-tubulin mAb (B-5-1-2, Merck), anti-ERK 2 rabbit polyclonal (C14, Santa Cruz Biotechnology), and Phospho-p44/42 MAPK rabbit polyclonal (Cell Signaling Technology); secondary antibodies used were HRP-conjugated goat anti-mouse IgG (BioLegend), rat anti-mouse IgG (Abcam) for detection of CD6 in MEM98 immunoprecipitates, and mouse anti-rabbit IgG (Santa Cruz Biotechnology). Membranes were developed with Amersham ECL Select/Prime Western Blotting Detection Reagent (Cytiva). Data were acquired using the ChemiDoc XRS+ System (Bio-Rad) and analyzed by Fiji [57 (link)].

Western blotting was performed by following the standard methods [19 (link)]. First, proteins were loaded on SDS/PAGE. After running, proteins in the gel were transferred on to PVDF membranes (Millipore, Bedford, MA, USA) by electrophoresis (Millipore, Billericay, Massachusetts). Then the membranes were blocked with 5% bovine serum albumin (BSA, Sigma–Aldrich, St. Louis, Missouri) for 1 h. Next, membranes were incubated with primary antibodies at 4°C overnight, which was followed by the secondary antibody incubation at room temperature for 1 h on the second day. The primary antibodies included anti-PTPN3 (1:1500, Abcam, Cambridge, MA, U.S.A.) and anti-GAPDH (1:1500, Santa Cruz Biotechnology). The secondary antibody was rabbit-anti-mouse IgG (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). ECL Chemiluminescence Detection System (Thermo Fisher Science, Rochester, New York) was used to detect the chemical signal. The intensities of individual protein bands were quantified by Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, U.S.A.). All procedures in the trial were repeated in triplicate.