Horseradish peroxidase coupled secondary antibody

Samples were diluted in loading buffer (final concentration: 60 mM Tris, 10% (v/v) glycerol, 5% (v/v) mercaptoethanol, 2% (w/v) SDS, 0.025% (w/v) bromophenol blue, pH 6.8), incubated for 5 minutes at 95°C and submitted to gel electrophoresis using AnykD gels (Biorad, Hercules, CA), and blotted onto PVDF membranes (BioRad). Membranes were washed in TBS-T and blocked in TBS containing 5% (w/v) dry milk powder, which was also used for antibody incubations. Incubation in primary antibody against α4 (1:100, sc-5591, Santa Cruz Biotechnology, Heidelberg, Germany), α7 (1:1000, ab23832, Abcam, Cambridge, UK), or β2 (1:1,000, a gift from Dr. Cecilia Gotti, which we have characterized previously [23 (link)]) was performed overnight at 4°C on parafilm in a humidified container, followed by 3 × 10 minute washes in TBS-T and 1 hour incubation at 20–22°C in horseradish peroxidase-coupled secondary antibody (1:1,000, Dako, Glostrup, Denmark). After thorough washing in TBS-T, enhanced chemiluminescence Western blotting detection reagents (Western Lightning ECL Pro, Perkin Elmer, Waltham, MA) were used for signal detection and protein bands were visualized using a Chemidoc XRS system with Quantity One software (Biorad). Mean optical densities of bands were measured and their corresponding background measurement subtracted.

The samples were mixed with a sample buffer (2 mmol L⁻¹ Tris-HCl, 0.2 mmol L⁻¹ EDTA, 20 mmol L⁻¹ DTT, 4% SDS, 10% glycerol, 0.04% bromophenol blue, pH 8.0). Equal amounts of protein were loaded into each lane and separated by 8–18% SDS–PAGE (Excel 8–18% gradient gel, Amersham).The proteins were then electroblotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). The membrane was blocked for 60 min at room temperature in a TS-buffer (10 mmol L⁻¹ Tris-Base, 0.9% NaCl, pH 7.4) containing 2% BSA, 1% skimmed-milk powder, and 0.1% Tween-20 before incubation with a primary antibody diluted in a similar buffer overnight (4°C). After treatment with a horseradish-peroxidase-coupled secondary antibody (Dako, Copenhagen, Denmark) for 90 min at room temperature, the membrane was repeatedly washed in TS-buffer with or without 0.05% Tween-20. The membrane was incubated with enhanced chemiluminescence reagent (ECL; Amersham) and visualized in an ImageQuant LAS 4000 image station. The samples that were to be compared were loaded on the same gel. The relative protein concentrations were quantified by the image station software.

Cells were lysed in
protein lysis buffer
(150 mM NaCl, 50 mM Tris, 1 mM EGTA, 1 mM Na₃VO₄, 10 mM NaF, 1% Triton X-100 and 1× proteinase inhibitor mix
from Roche), and protein concentration was determined with a Bradford
protein assay from BioRad following the manufacturer′s instructions.
For analysis of SLC26A1 and SLC41A, 30 and 80 μg, respectively,
of total protein per lane were separated by SDS polyacrylamide gel
electrophoresis and subsequently electroblotted onto PVDF membranes.
Membranes were blocked in 5% milk powder for 1 h and subsequently
incubated with the following primary antibodies overnight at 4 °C:
rabbit polyclonal anti-SLC26A1/SAT1, 1:200, Proteintech, rabbit monoclonal
anti-SLC4A1, 1:100, Cell Signaling Technology and mouse monoclonal
anti-β-actin, 1:3000, Sigma. After washing with Tris-buffered
saline with 0.1% Tween 20, membranes were incubated in horseradish
peroxidase-coupled secondary antibodies (Agilent) for 1 h at room
temperature. Luminescence signals were developed with Clarity Western
ECL substrate (BioRad) and recorded on X-ray film.

Efficiencies of conditional GRK depletion in BMDCs were determined by immunoblot analysis. BMDCs were lysed (50 mM Tris, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 0.5% IGEPAL^® CA-630, 1% Triton™ X-100, 1x cOmplete™ protease inhibitor cocktail (Roche)) and lysates loaded on 12% polyacrylamide gels for electrophoresis under reducing conditions (SDS-PAGE). Separated proteins were immunoblotted on PVDF membrane and unspecific binding blocked with 5% milk powder in Tris-buffered saline with 0.1% Tween^® 20 (TBS-T). Membranes were stained overnight at 4°C with primary antibodies (listed in Supplementary Table 1) and actin as loading control and, after three wash steps with TBS-T, stained with horseradish peroxidase-coupled secondary antibodies (Dako). Chemiluminescence was detected using Clarity™ Western ECL substrate and a ChemiDoc™ Imaging System (both BioRad).

Cells were lysed in NP40 lysis buffer (1% NP40, 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA). Following the determination of protein concentrations by Bradford assay (Bio-Rad), the equivalent of 20 μg of total protein (or 60 μg for analysis of m42) was loaded per well on 6–8% SDS-polyacrylamide gels (or 13% for analysis of m42). Antibodies used for probing the blots were CD45 (69/CD45; BD); GAPDH (14C10; Cell Signaling); m42 (m42.02); IE1 (Croma101); E1 (Croma103), M57 (M57.02). Antibodies directed against MCMV proteins were generated and provided by CAPRI (Rijeka, Croatia). Horseradish peroxidase-coupled secondary antibodies (Dako) were used at a 1:5000 dilution. Signals were detected with an LAS-3000 imager following treatment with the ECL Select substrate (GE Healthcare). Images were processed using Adobe Photoshop CS4.