Rabbit anti snap tag

For hepatic LHBS staining, paraffin-embedded tissues were pre-warmed at 65°C for 15 min and treated with xylene for paraffin removal. Antigen retrieval was performed by boiling the samples in a tissue pretreatment buffer solution (Invitrogen) for 30 min. The slides were then incubated with mouse anti-preS1 for 1 hr and assessed using the EnVision+ System-HRP kit (DAKO, Carpintena, CA, USA). For double immunofluorescence staining, the cells were fixed and stained with the indicated primary antibodies for 1 hr, followed by secondary antibodies conjugated with Alexa-488, Alexa-594, or Alexa-647 (Molecular Probes). The cells were mounted with mounting medium that contained DAPI. The images were acquired with a Zeiss LSM780 confocal microscope or an automated Leica DMI6000 inverted microscope equipped with an HCX PL FL 20x/NA0.4 objective and an EMCCD camera (Andor Luca R, Belfast, UK) as indicated. The following primary antibodies were used in this study: mouse anti-preS1 (a gift from Ningshao Xia, Xiamen University, China), rabbit anti-SNAP tag (P9310S, New England Biolabs, MA, USA), rabbit anti-STIM1 (PA5-23623, Thermo Scientific, IL, USA), and mouse anti-γ-tubulin (sc-17787, Santa Cruz, Dallas, Texas, USA).

HEK293 cells were plated in 12 or 24-well plates. After 24 h, cells were transfected using Lipofectamine 2000 according to the manufacturer’s instructions. Protein lysates were obtained using urea lysis buffer (0.5% NP-40, 2% SDS, 75 mM NaCl, 88 mM Tris/HCl, 4.5 M urea, 10% β-mercaptoethanol, 10% glycerol, pH 7.4). Lysates were sonicated and analyzed by 7.5, 10, or 4–20 % Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) and transferred to PVDF membranes using the Trans-Blot Turbo system (Bio-Rad). After blocking with 5% milk in TBS-T, membranes were incubated with primary antibodies in blocking buffer: rabbit anti-GAPDH (1:8000; Cell Signaling Technology #2118), rabbit anti-DVL2 (1:1000; Cell Signalling Technology #3216), rabbit-anti-P-S648 FZD₆ antibody (1:500; custom made), and rabbit anti-SNAP tag (1:1000, New England Biolabs #P9310S) overnight at 4 °C. The anti-P-S648 antibody was raised on a service basis by Moravian Biotechnology and validated previously^{52 (link)}. Proteins were detected with horseradish peroxidase-conjugated secondary antibody (1:10000; goat anti-rabbit (Thermo Fisher Scientific #31460)) and Clarity Western ECL Blotting Substrate (Bio-Rad). All uncropped immunoblots can be found in the Supplementary Figure 11.

Samples were fractioned by SDS–PAGE on 8% gels under reducing conditions, immunoblotted onto nitrocellulose membranes (GE Healthcare) and bands detected by enhanced chemiluminescence (GE Healthcare) onto films developed on a Xograph Compact X5 processor.
Antibodies were primaries rabbit anti-β-arrestin-1/2 (D24H9, Cell Signaling, 1/1000), rabbit anti-SNAP tag (New England Biolabs, 1/500), mouse anti-α-tubulin (T5168, Sigma, 1/1000), rabbit anti-β-actin (4970, Cell Signaling, 1/1000), and mouse anti-GAPDH (6C5, Merck, 1/10,000); and IgG-HRP secondaries (Santa Cruz Biotechnology).