Re blot plus mild solution

Proteins were separated on a 10% SDS-PAGE gel and immunoblots were performed following the protocol from Green and Sambrook (2012 ). Protein was transferred to a nitrocellulose membrane, or a polyvinylidene fluoride (PVDF) membrane if fluorescent secondary antibodies were used. After blocking membranes overnight, membranes were incubated with GFP (Invitrogen, cat. no. A6455) or RbcL (Agrisera, cat. no. AS03037) antibodies, or streptavidin-HRP (Life Technologies, cat. no. R960-25). Membranes probed for GFP or RbcL were then incubated with a secondary antibody conjugated to HRP or AlexaFluor 488 (Thermo Fisher, cat. no. A-11008 or cat. no. 31460). Membranes were visualized using chemiluminescence after exposure to the Clarity Western ECL substrate (Bio-Rad) or fluorescence. If necessary, blots were stripped using ReBlot Plus Mild Solution (Millipore).

Cells were collected by trypsinization, washed with ice-cold in PBS and incubated on ice in extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8, 0.5% NP40, 10 mM benzamide hydrochloride, 20 mM NaF, 20 mM β-glycerophosphate, 1 mM Na₃VO₄, 5 mM MgCl₂, 125 U/ml Benzonase nuclease (Novagen) and 1x Protease inhibitor cocktail (Sigma-Aldrich) for at least 30 min. Supernatant was collected as whole cell extract. For immunoprecipitation, the obtained cell extracts were pre-cleared with uncrosslinked Affiprep Protein-A beads (Bio-Rad) for at least 1 h at 4 ^oC. The pre-cleared lysate was subsequently incubated with antibody-crosslinked Affiprep Protein-A beads overnight at 4 ^oC. After extensive washing in extraction buffer, immune complexes were eluted from the beads in NuPAGE LDS sample buffer (ThermoFisher Scientific) supplemented with 12 mM DTT by heating the sample at 85 °C for 5 min. Western blotting was performed following standard protocol. Primary and secondary antibodies were applied at the dilutions described in the Supplemental Methods. Where indicated, the membrane was treated with Re-Blot Plus Mild Solution (Millipore) before incubating with another antibody.