V src

Protein extraction from both yeast and mammalian cells was carried out using methods previously described (Mollapour et al., 2010 (link)). For immunoprecipitation, mammalian cell lysates were incubated with anti-FLAG or anti-HA antibody conjugated agarose beads (Sigma) for 2 h at 4°C. Immunopellets were washed 4 times with fresh lysis buffer (20mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 0.1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) and eluted in 5x Laemmli buffer. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Co-immunoprecipitated proteins were detected by immunoblotting with antibodies recognizing FLAG, 6x-His (ThermoFisher Scientific), Hsp90-835-16F1, GAPDH, p23 (ENZO Life Sciences), Tsc1, FLCN, GR, Myc, V5, GAPDH, Hsp90α, FNIP2, c-Src (Cell Signalling), phospho-tyrosine, v-Src (Millipore), FNIP1, FNIP2 (NCI), FNIP1 (antibodies-online.com), Aha1 (StressMarq Biosciences), HA (Roche). Secondary antibodies raised against mouse, rabbit, and rat (Cell Signaling) and goat (Santa Cruz Biotechnology) were used (See Key resources table).

For pulldown, yeast cell lysates were incubated with Ni-NTA agarose (Qiagen) for 2 h 4°C or anti-FLAG antibody conjugated agarose beads (Sigma) for 2 h at 4°C. Protein bound to beads was washed with fresh extraction buffer 4 times and eluted in 5x Laemmli buffer. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. IP of protein from mammalian cells was performed as above with anti-FLAG conjugated agarose beads.
Co-precipitated proteins and inputs were detected by immunoblotting with antibodies recognizing FLAG, 6x-His (ThermoFisher Scientific), phos-(Ser/Thr)-Phe, GAPDH, HOP, hCdc37, Ulk1, myc-tag, LC3B (Cell Signaling), phosphoserine, phospho-threonine, FLAG (Sigma-Aldrich), GFP (BioLegend) tubulin (Abcam) phosphotyrosine, v-Src (Millipore), p23 (ENZO Life Sciences), hAha1 (StressMarq Biosciences), Sti1^Hop (a kind gift from Dr. Daniel C. Masison, NCI, USA), Cdc37^p50 and yAha1 (a kind gift from Dr Len Neckers, NCI, USA), Sba1 (Institute of Cancer Research, UK). Secondary antibodies raised against mouse, rabbit, and rat (Cell Signaling) and anti-mouse Alexa Fluor 488 (ThermoFisher were also used (See Key Re-sources Table).