Rabbit anti flag m2

HEK293T cells (2 × 10⁶) were seeded in 6 cm-dish and co-transfected with HA-tagged WT-CCR6 or various mutant CCR6 and β-arrestin 1-flag, β-arrestin 2-flag, or β-arrestin 2-flag-GFP for 24 h. Cells were trypsinized, resuspended at a density of ~1 × 10⁷ cells/ml in fasting medium, stimulated with 100 ng/ml CCL20 for the indicated time, incubated in a dry ice/ethanol bath to stop reaction and lysed with lysis buffer for 30–60 min on ice. After centrifugation at 12,000 × g for 10 min, supernatants (~1 mg) were pre-cleared with protein A agarose beads (Millipore, Billerica, MA, USA) for 1 h at 4°C. Pre-cleared lysates were incubated with anti-HA affinity gel (Sigma-Aldrich) for 3 h at 4°C and the immunoprecipitates were washed, denatured with Laemmli buffer for 30 min at 50–55°C and subjected to 9% SDS-PAGE, followed by western blotting with rabbit anti-flag M2 (Sigma-Aldrich) or Rat anti-HA 3F10 (Roche).

Our studies employed the following primary antibodies: Rabbit monoclonal anti-Engrailed (Monoclonal Antibody Facility, Institute of Neuroscience, University of Oregon), mouse polyclonal anti-Prox1 (AngioBio, 11-002P), mouse anti-FLAG M1 (prepared in-house, 1:5,000), rabbit anti-FLAG M2 (Sigma, F7425, 1:1,000), rabbit anti-GFP (which also detects YFP) (Thermo Fisher Scientific A11122, 1:5,000), mouse anti-PKA-C (BD Biosciences, 610980, 1:5,000), mouse anti-PKA-R (BD Biosciences, 610609, 1:500), mouse anti Arl13b (Antibodies, 75–287, 1:1,000), rabbit anti-RFP (Thermo Fisher Scientific, R10367, 1:1,000), rabbit anti-CREB (Cell Signaling Technology, 9197S, 1:1,000), and rabbit anti phospho-CREB (Cell Signaling Technology, 9198S, 1:1,000). For chemiluminescent western blots, HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Promega and used at 1:20,000. For infrared western blots, IR680- and IR800-conjguated secondary antibodies (LiCor) were used at 1:20,000. For immunofluorescence, AlexaFluor-conjugated secondary antibodies were obtained from Thermo Fisher Scientific and used at 1:1,000. M1 FLAG affinity resin and M1 FLAG-Alexa 647 conjugates were prepared in house.