Epidermal lysates were prepared by mechanical homogenization in RIPA buffer
supplemented with HALT phosphatase inhibitor cocktail (Thermo Scientific, IL) and Complete
mini protease inhibitor cocktail (Roche, IN) followed by protein quantification using
standard Bradford method with Bio-Rad Protein Assay dye reagent (Bio-Rad Laboratories,
CA). For immunoprecipitation, 500 μg of pre-cleared protein lysate was incubated
with 5 μg of primary mouse 2t2 antibody overnight at 4°C followed by binding
to Protein G Sepharose beads (GE Healthcare, Sweden). Beads were centrifuged, washed, and
the pellet dissolved in Laemmli buffer. Supernatants were separated on 4-20% or 12%
gradient SDS-polyacrylamide gels followed by transfer to Immobilon-P membranes (Millipore,
MA). Antibody details are listed inSupplementary Material . Detection was carried out with SuperSignal West Pico or
Femto chemiluminescent substrates (Thermo Scientific, IL).
supplemented with HALT phosphatase inhibitor cocktail (Thermo Scientific, IL) and Complete
mini protease inhibitor cocktail (Roche, IN) followed by protein quantification using
standard Bradford method with Bio-Rad Protein Assay dye reagent (Bio-Rad Laboratories,
CA). For immunoprecipitation, 500 μg of pre-cleared protein lysate was incubated
with 5 μg of primary mouse 2t2 antibody overnight at 4°C followed by binding
to Protein G Sepharose beads (GE Healthcare, Sweden). Beads were centrifuged, washed, and
the pellet dissolved in Laemmli buffer. Supernatants were separated on 4-20% or 12%
gradient SDS-polyacrylamide gels followed by transfer to Immobilon-P membranes (Millipore,
MA). Antibody details are listed in
Femto chemiluminescent substrates (Thermo Scientific, IL).