Anti β actin ab6276

After 48-hour exposure to different glucose concentrations and 6-hour oxygen tensions, cells were washed with PBS, collected by centrifugation, and lysed in a high-salt buffer containing 50 mmol/L Tris-HCl (pH 7.4), 500 mmol/L NaCl, 0.2% NP-40, 20% glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L beta-mercaptoethanol, and a protease inhibitor mix (cOmplete, Mini; Roche Applied Science). The lysates were then cleared by centrifugation for 30 min at 20,000 g at 4°C. The whole-cell extracts were separated by SDS-PAGE and blotted onto nitrocellulose membranes. After blocking in TBS buffer (50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl) containing 5% nonfat milk, the membranes were incubated with anti-FLAG M2 (F3165, Sigma-Aldrich) or anti-β-actin (ab6276, Abcam) antibodies in TBS buffer containing 1% nonfat milk. After several washes with TBS buffer containing 0.5% Tween 20, the membranes were incubated with anti-mouse or anti-rabbit IgG-horseradish peroxidase conjugate (Amersham Biosciences Corp.) in TBS buffer containing 1% nonfat milk. After several washes, proteins were visualized using enhanced chemiluminescence (Amersham Biosciences Corp.) according to the manufacturer's recommendations.

Antibody arrays were analysed using Phospho Explorer Antibody Arrays (Full Moon Biosystems, Sunnyvale, CA, USA). Western blots were performed for anti‐RRN3 pSer649 (ab138651) and anti‐β‐actin (ab6276), both from Abcam, Cambridge, UK; anti‐stathmin pSer38 (4191), anti‐p90RSK pThr573 (9346), anti‐Bad pSer112 (9291), anti‐RSK1/2/3 (9355), anti‐Bad (9292) and anti‐stathmin (3352), all from Cell Signalling Technology, Cambridge, UK; anti‐α‐tubulin (T9026) and anti‐MELK (HPA017214), all from Sigma‐Aldrich, St Louis, MO, USA; anti‐RRN3 (sc‐133978), from Santa Cruz Biotechnology, Dallas, TX, USA; and anti‐MELK (NBP1‐19598), from Novus Biologicals, Littleton, CO, USA. IF was performed for α‐tubulin (DM1A, Sigma) and IHC for MELK (NBP1‐19598, Novus Biologicals, Littleton, CO, USA) and CC3 (9664, Cell Signalling Technology). qRT–PCRs were performed on an ABI PRISM 7900 HT Sequence Detection System. Relative gene expression was calculated according to the ∆∆Ct method; HPRT was used as housekeeping gene.

Total protein was extracted from the testes of rats and co-cultured Sertoli–germ cells exposed to different concentrations of MC-LR with or without RES. Samples containing 30 µg of protein underwent electrophoresis with a Bio-Rad electrophoresis apparatus (Bio-Rad, Hercules, CA, USA), were separated by 12% SDS-PAGE, and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked in tris buffered saline with tween (TBST) containing 5% BSA at 23 °C for two hours, and immunoblotted using primary anti-Ku70 (sc-17789, Santa Cruz Biotechnology, Boston, CA, USA), anti-SIRT1 (ab110304), anti-p53 (ab131442), anti-p53 (acetyl K381, ab61241), anti-cleaved-caspase-3 (ab2302), anti-Bax (ab32503), anti-Bcl-2 (ab7973), and anti-β-actin (ab6276) (Abcam, Cambridge, UK). An enhanced chemiluminescence detection kit (Beijing ComWin Biotech Co., Ltd., Beijing, China) was used to analyze the protein bands. The intensity of the bands was quantified using the Bio-Rad Quantity One software.

Adalimumab (Humira) was purchased from AbbVie Inc. (North Chicago, IL, USA). It was first dissolved in the N.S at a final concentration of 1 mg/mL and then injected intraperitoneally (i.p.) at a dose of 5 mg/kg four times a week for 9 weeks [12 (link)]. Anti-Iba1 (ab178847, 1:200 for immunofluorescence), anti-nuclear factor kappa B (NF-kB) p65 (p65, ab16502, 1:1000 for western blotting), anti-phospho-Ser536-NF-kB p65 (p-p65, ab-28856, 1:800 for western blotting), anti-STAT3 (ab68153, 1:1000 for western blotting) and anti-β-actin (ab6276, 1:1000 for western blotting) antibodies were purchased from Abcam Inc. (Shanghai, China). Commercial kits for detecting the inflammatory factors TNFα (kt30484), IL-6 (kt30490), IL-12 (kt30428), IFNγ (kt77521), IL-1β (kt45331), IL-10 (kt30495) and IL-4 (kt30491) were purchased from MSK Company (Wuhan, China). ELISA kits for measuring SOD (A001-3-2), MDA (A003-1-2), GSH-Px (A005-1-2), and CAT (A007-1-1) levels were purchased from Nanjing Jianchen Company (Nanjing, China).

The following antibodies were used: LACC1 E-12 (anti-FAMIN; sc-376231), E-7 (sc-374553), H-6 (sc-376064) from Santa Cruz; anti-β actin (ab6276) and anti-PMP70 (ab3421) from Abcam; anti-β-tubulin (2128), anti-calnexin (2679), anti-calreticulin (12238), anti-catalase (12980), anti-caveolin-2 (8522), anti-CENP-A (2186), anti-COX-IV (4850), anti-EEA1 (3288), anti-fibrillarin (2639), anti-histoneH3 (4499), anti-LAMP1 (9091), anti-LC3B (2868), anti-NUP98 (2598), anti-PDI (2446), anti-Rab5 (3547) and anti-syntaxin-6 (2869) from Cell Signaling; anti-ABCD3 (HPA032027) from Sigma Aldrich.