Anti β actin monoclonal antibody

Proteins were extracted by homogenization of the spleen on days 2, 4, and 14 in tissue protein extraction reagent (Pierce, Rockford, IL, USA) to determine the levels of SOCS3 protein, as described previously [19 (link)]. Monoclonal anti-β-actin antibody was obtained from Santa Cruz (St. Louis, MO, USA). Anti-mouse SOCS3 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Peroxidase-conjugated goat immunoglobulin G was purchased from Santa Cruz.

Monoclonal anti-phospho-specific-FAK and anti-FAK antibodies were obtained from Becton Dickinson (Heidelberg, Germany). Anti-Sirt1 and anti-CXCR4 (CXC-Motiv-Chemokinreceptor 4) antibodies were purchased from Abcam PLC (Cambridge, UK). Anti-phospho-specific p65 (NF-κB) and anti-phospho-specific p50 (NF-κB) antibodies were obtained from Cell Technology (Beverly, MA, USA). Anti-active caspase 3, anti-MMP-9 and anti-MMP-13 antibodies were obtained from R&D Systems (Heidelberg, Germany). Monoclonal anti-β1-Integrin and anti-β-actin antibodies were purchased from Sigma-Aldrich Chemie (Munich, Germany). Monoclonal anti-β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alkaline phosphatase–linked sheep anti-mouse and sheep anti-rabbit secondary antibodies for immunoblotting were purchased from EMD Millipore (Schwalbach, Germany). Anti-Ki-67 and secondary antibodies used for fluorescence labeling were obtained from Dianova (Hamburg, Germany). All antibodies were used at concentrations recommended by the manufacturers.

The protein extraction and immunoblotting was performed generally following the previous reports [26] (link). The hippocampus CA1 tissues were collected and lysed in ice-cold lysis buffer containing 55 mM Tris–Cl, 145 mM NaCl, 0.01 mM NaN₃, 100 μg/ml phenylmethyl sulfonyl fluoride, 1 μg/ml aprotinin, 1% Triton X-100 and proteinase inhibitor cocktail. Cytoplasmic and nuclear protein were extracted and separated with SDS - polyacrylamide gel (7.5%) electrophoresis followed by blotting to a nitrocellulose membrane. The blots were incubated overnight at 4°C with the primary antibodies as following: polyclonal anti-MeCP2 primary antibody (1∶1000; Cell signaling), polyclonal anti-phosphorylated MeCP2 (serine 421) antibody (1∶1000; ECM Biosciences) and monoclonal anti-β-actin antibody (1∶2000; Santa Cruz Biotechnology). After extensive wash, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibody (1∶10,000; Jackson ImmunoResearch Laboratories Inc.). The immunoreactivity was detected using enhanced chemiluminescence (ECL Advance Kit; Amersham Biosciences). The intensity of the bands was captured digitally and analyzed quantitatively with ImageJ software. The immunoreactivity of all proteins was normalized to that of β-actin.

The tissue samples from patients were further taking for immunoblotting. The tissue samples were lysed in extraction buffer (150 mM NaCl, 10 mM Tris, 5 mM EDTA, 1% Triton X-100, 5% glycerol, and 0.1% SDS, pH 7.2), then were centrifuged at 22,000 rpm, 4°C for 30 min. The supernatants were collected, and the protein concentration was determined according to BCA protocol. Equal amount of proteins from each patient were applied for the immunoblotting experiments. 40 μg proteins per patients were analyzed on 12.5% SDS-PAGE, and transferred on PVDF membrane (transfer buffer: 25 mM Tris, 192 mM glycine, 20% methanol, pH8.3) by the Bio-Rad Semidry apparatus with a constant current of 300 mA for 1 hour or 30 min according to the size of the protein. Afterwards, the membrane was blocked in blocking buffer (1 × PBS, 0.5% Tween-20 with 5% nonfat milk) for 2 hours, and then further blotted with the primary antibody (monoclonal anti-S100A12 antibody, 1:500 dilution, Abcam; monoclonal anti-AMACR antibody, 1:1000 dilution, Abcam; monoclonal anti-β-actin antibody, 1:1000 dilution, Santa Cruz) in 4°C overnight and HRP conjugated secondary antibody in room temperature for 2 hrs. Afterwards, the membrane was incubated with Chemi-luminescent Detection Reagent (Pierce) for 5 min, and then was exposed to X-ray film. All of the above mentioned experiments were independently repeated 3 times.

Proteins were extracted by homogenization of the kidney at 4, 24, and 48 h after LPS-injection and TECs in tissue protein extraction reagent (Pierce, Rockford, IL, USA) to determine Bax, as described elsewhere [47 (link)]. Monoclonal anti-β-actin antibody was obtained from Santa Cruz Biotechnology (St. Louis, MO, USA). Anti-mouse Bax antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Peroxidase-conjugated goat immunoglobulin G was purchased from Santa Cruz Biotechnology.