Horseradish peroxidase conjugated goat anti rabbit secondary antibody

Log phase cultures were resuspended to 6 × 10⁸ cells per mL in reduced osmolarity PBS solution (“mPBS” = 7.2 mM NaCl, 5.4 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). 100 μL of 10% paraformaldehyde solution (in mPBS) and 1 μL of 5% glutaraldehyde solution (in H₂O) were added to 400 μL of cell suspension. Each mixture was spotted on a poly-lysine coated slide. Fixation proceeded for 30 min at RT. After rinsing with mPBS, cells were permeabilized with 0.025% Triton X-100 for 10 min and washed. Cells were blocked for 1 hr at RT with 4% BSA in mPBS, then probed with a 1:1000 final concentration of anti-FLAG antibody (in 4% BSA, Sigma, St. Louis, MO) for 1 hr at RT and subsequently washed 2× for 5 min and 1× for 10 min with mPBS. Cells were then probed with a 1:2000 final concentration of secondary antibody (in 4% BSA, Alexa Fluor 488-conjugated donkey anti-rabbit IgG; Jackson ImmunoResearch, Westgrove, PA) for 30 min at RT and subsequently washed as before. SlowFade Gold antifade reagent (Invitrogen) was added to the slide and the cells were visualized with a 100× objective lens. Western blot was performed according to standard protocols, using anti-FLAG antibody described above, and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Thermo Scientific, Waltham, MA).

SARS-CoV-2 super stable hexapro (6 P) spike trimer was incubated either with hACE2 ectodomain (Acro biosystem, 1:10 molar ratio) or an Nb (1:8 molar ratio) overnight at room temperature. Proteins were then digested with proteinase K (PK, 1:50 enzyme to substrate ratio) for 15 min and 60 min at room temperature. PK was inactivated by mixing with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer and heating at 98 °C for 10 min. Inactivated samples were run on a 4–12% Bis–Tris gel (Bolt) before being stained with a Sypro Ruby stain or subject to western blot analysis. For western blot, anti-S2 SARS-CoV-2 polyclonal antibodies (Sino biologics, Cat# 40590-T62, 1:2000 dilution) were used as the primary antibody at 4 °C overnight. A horse radish peroxidase-conjugated goat anti-rabbit secondary antibody was used at 1:5000 dilution (Thermo Fisher, Cat# 31460) for 1 h at room temperature. ECL substrate (Bio-rad) was used to develop S2 signals which were visualized by the Bio-rad Imager. The experiments were repeated four times.

Reagents were purchased from the following suppliers: GAPDH and catalase antibodies from Cell Signaling Technologies; horseradish peroxidase–conjugated goat anti-rabbit secondary antibody from Thermo Fisher Scientific; bile acid standards from CDN Isotopes, Cambridge Isotope Labs, and Avanti Polar Lipids; MS-grade acetonitrile from Honeywell; TRI Reagent from Molecular Research Center, Inc.; ¹⁴C-palmitic acid from American Radiolabeled Chemicals, and etomoxir from Tocris Bioscience. The NUDT7 antibody was generated as described previously (18 (link)). All other chemicals were of analytical grade or better and were purchased from Sigma-Aldrich or Thermo Fisher Scientific, unless stated otherwise.

Mice ears were injected with chemically-modified FFAR2 and control siRNAs followed by application with 2% PA and UVB exposure for 3 days. On third day mice ears were cut, homogenized and then lysed with RIPA buffer (Thermo Fisher Scientific). Cell lysates (30 µg) were subjected to 10% SDS-PAGE gel, which were then transferred to a poly(vinylidene fluoride) (PVDF) membrane (Sigma) and blocked with 5% (w/v) nonfat milk before incubation overnight with primary antibodies to FFAR2 Rabbit PolyAb (Proteintech, Rosemont, IL, USA) at 4 °C or β-actin (1:1,000; Cusabio Technology, Houston, TX, USA). This was followed by treatment with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000) (Thermo Fisher Scientific) for 1 h. Protein bands were detected with a chemiluminescent detection reagent (Thermo Fisher Scientific) and Omega Lum C Imaging System (Gel Co., San Francisco, CA, USA). Protein bands were conducted using ImageJ software (https://imagej.nih.gov/ij/; Version 1.53e).

Total cell lysates were obtained using RIPA buffer, and extracted using centrifugation at 18,000 × g at 4 °C for 25 min. The proteins for Western blot were denatured by 5× Tris-acetate sample buffer at 95 °C for 5 min. The protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich). The samples (20 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Following blocking with 5% nonfat milk for 1 h in Tris-buffered saline with 0.05% Tween-20 buffer (TBST) at room temperature, the membranes were incubated overnight with primary antibody at 4 °C. Following TBST washing, the membranes were incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific). The protein bands were detected and visualized using an electrochemiluminescence system (Amersham Imager 600; GE Healthcare Bio-Sciences, Piscataway, NJ, USA).

Pancreas tissue (30 mg) was homogenized in 300 µL phosphate-buffered saline (pH 7.4) containing Halt Protease and Phosphatase Inhibitor Cocktail (catalog number 78440, Thermo Fisher Scientific) and 30 µg total protein of the cleared lysate was loaded per well. Mouse CTRL was detected using a rabbit polyclonal antibody raised against a synthetic peptide that corresponds to amino-acids 66–115 of human CTRL (catalog number AV33864, MilliporeSigma, St. Louis, MO). This sequence is 84% identical to the corresponding mouse CTRL region. The antibody was used at a final dilution of 1:1,000. Rabbit monoclonal antibody against p44/42 MAPK (ERK1/2) (137F5) was used at a final dilution of 1:500 (catalog number 4695, Cell Signaling Technology, Danvers, MA). The horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was used at a dilution of 1:20,000 (catalog number 31460, Thermo Fisher Scientific).