Hrp conjugated donkey anti goat igg

Lysates and immunoprecipitates were loaded onto NuPAGE™ 4-12%, Bis-Tris, 1.5 mm gels (Thermo Fisher Scientific) and resolved. Gels were wet blotted onto polyvinylidene fluoride (PVDF) membranes and subsequently blocked with 5% BSA in TBST for 1 h at ambient temperature. Blots were incubated with goat anti-Arap1 IgG (1:2000, Abcam, ab5912) and rabbit anti-β-actin IgG (1:4000, Cell Signaling Technology, 4970S) overnight at 4°C. After incubation with horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (1:4000, Abcam, ab97110) and HRP-conjugated goat anti-rabbit IgG (1:4000, Cell Signaling Technology, 7074P2) for 1 h at ambient temperature, bands were detected with enhanced chemiluminescence western blotting detection reagents (Amersham). All antibodies used in this study are commercially available and were formerly validated by their respective companies. Catalogue numbers, dilutions and PMIDs of previous publications using all primary antibodies are summarized in Table S1.

Freshly isolated mouse livers were lysed with a lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 0.1% SDS with protease inhibitors (1:100; Thermo Fisher Scientific). Protein samples were separated by SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was incubated with primary goat anti-mouse CLEC4F (R&D Systems) followed by HRP-conjugated donkey anti-goat IgG (Abcam).