Rabbit anti actin a2066

The following primary antibodies were diluted in TBST plus 5% nonfat dry milk for western blot: rabbit anti-mGBP-2 (1851 [21 (link),22 (link)]; 1:20,000), rabbit anti-actin (A2066; 1:3000) (Sigma), rabbit anti-α-tubulin (GTX102078; 1:10,000) (GeneTex, Irvine, CA, USA), mouse anti-GAPDH (60004; 1:20,000) (Proteintech, Rosemont, IL, USA). The above antibodies were incubated with membrane for 1 h at RT. The rabbit anti-RhoA (2117T; 1:4000) (Cell Signaling, Danvers, MA, USA), mouse anti-Rac1 (610650; 1:4000) (BD Biosciences, San Jose, CA, USA), mouse anti-CDC42 (610928; 1:250) (BD Biosciences), rabbit anti-Akt (9272; 1:3000) (Cell Signaling), and rabbit monoclonal anti-phosphoAkt (4060 (ser 473); 1:4000) (Cell Signaling) were incubated with membrane overnight at 4 °C. Hrp-conjugated goat anti-mouse immunoglobulin (1:8000) or goat anti-rabbit immunoglobulin (1:6000) (Jackson ImmunoResearch, West Grove, PA, USA) were diluted in TBST containing 5% w/v nonfat dry milk and incubated with the membranes for 1 h at RT.

Lipopolysaccharide (LPS) from Escherichia coli O111:B4, lactic acid (LA), sodium lactate (NaL), L-arginine hydrochloride (Arg-HCl), and L-arginine methyl ester dihydrochloride (Arg-ME) were purchased from Sigma Aldrich (Taufkirchen, Germany), whereas interferon γ (IFN-γ) and 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino] diazen-1-ium-1,2-iolate (DETA-NO) were obtained from Invitrogen (Darmstadt, Germany) and Cayman Chemical (Ann Harbor, MI), respectively. Hydrochloric acid (HCl) was purchased from Fisher Chemical (Schwerte, Germany). RPMI 1640, DMEM and PBS were purchased from Gibco (Darmstadt, Germany). Immunoblotting was carried out using the following antibodies: rabbit anti-Actin (A2066; Sigma Aldrich), mouse anti-HSP90α/β (sc-7947; Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-NOS2 (ADI-KAS-NO001; Enzo Life Sciences, Lörrach, Germany) and mouse anti-Arginase 1 (sc-166920; Santa Cruz). Either swine anti-rabbit HRP (P0399, Dako, Hamburg, Germany) or goat anti-mouse HRP (P0447, Agilent) were used as secondary antibodies.

The frozen tissue was pulverized with a metal cone using MULTI-BEADS SHOCKER MB1200 and dissolved in 1% SDS buffer (62.5 mM Tris/HCl [pH 6.8], 1% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.02% bromophenol blue, cOmplete protease inhibitor, and Phosstop). The samples were sonicated for 10 s at three times. After centrifugation (10,000 g, 20°C, 3 min), supernatants were collected in new tubes. Proteins were quantified by BCA analysis (Thermo Fisher Scientific). 10–20 μg proteins were applied to SDS-PAGE and subsequently electrotransferred onto PVDF membranes (Merck Millipore). Immunoblotting was performed using primary and secondary antibodies: rabbit anti-Actin (A2066; 1:3000; Merck Millipore), rabbit anti-delta 4-desaturase, sphingolipid 1 (DEGS1; PA5-42741; 1:1000 dilution; Thermo Fisher Scientific), and HRP-conjugated anti-rabbit IgG F(ab’)₂ fragment (ab6721, 1:5000 dilution; Abcam, Cambridge, UK). ECL Western blotting substrate (Bio-Rad, Berkeley, CA, USA) was used for labeling.

Liver or kidney extracted proteins (25 μg homogenate) were resolved on 10% polyacrylamide gels by SDS-PAGE electrophoresis, transferred to a nitrocellulose membrane, and then incubated with a GR-specific antibody ((rabbit anti-GR (N-terminal, M-20; Santa Cruz, CA, USA), rabbit anti-GR (C-terminal amino-acids 755–771, PA1-516, Thermo Fischer Scientific)). sEH expression was analysed in 30 µg of protein lysate (rabbit anti-sEH, Cayman Chemicals, 10010146). Immunoreactive bands were visualized by chemiluminescence (ECL kit; Amersham Biosciences, 152 Little Chalfont, UK). An anti-actin monoclonal (rabbit anti-actin (A2066, Merck)) or a mouse anti-GAPDH antibody (Merck) were used to verify equal protein loading between samples.