Interactome of endogenously HA-tagged GABARAPL2

Version 1

We selected GABARAPL2endoHA cells for a proof-of-principle immunoprecipitation (IP) followed by mass spectrometric (MS) analysis to identify new binding partner candidates of a hATG8 family member at endogenous levels. To distinguish between candidates that bind preferentially to PE-conjugated versus unconjugated GABARAPL2 we treated stable isotope labeling with amino acids in cell culture (SILAC)-labeled GABARAPL2endoHA cells with Torin1 and BafA1 (light) or ATG7 inhibitor (heavy).

Frozen cell pellets from 4x15 cm cell culture plates were lysed in Glycerol buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.5 % Triton-X-100, 10 % Glycerol, 1x protease inhibitor, 1x phosphatase inhibitor) for 30 min at 4° C with end-over-end rotation. Lysates were cleared from cell debris by centrifugation prior to adjustment of protein concentrations between the samples and overnight immunoprecipitation at 4° C with pre-equilibrated anti-HA-agarose (Sigma). Agarose beads were washed five ...