Erbb2

HEK293 cells were grown in 6-well plates to 50% confluency and transfected with XtremeGENE 9 transfection reagent according to the provided protocol. Plasmids encoding FLAG-tagged HSP90 mutant constructs were co-transfected with plasmids encoding each client protein and allowed to express for 18 hours. Cells were lysed with TGNET buffer (50 mM Tris HCl, pH 7.5, 5% Glycerol, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100) complete with protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche), then centrifuged at maximum speed for 15 minutes at 4°C. After BCA protein assay, 40 μg of protein were used as input lysate. For immunoprecipitation, 700 μg of protein from remaining lysates were added to 40 μl of anti-FLAG Beads (Sigma-Aldrich, A2220) and incubated with rotation for 2 hours at 4°C. The pull-down beads were washed 4 times with TGNET buffer. The proteins were eluted with 30 μl of 2X SDS sample buffer by boiling at 95°C for 5 min. Subsequently, samples were subjected to SDS-PAGE followed by Western blotting. The following antibodies were used in this report: FLAG (Sigma-Aldrich, A8592), HA (Rockland, #600-401-974), ERBB2 (Thermo Scientific, MS-730-P1), pERBB2 (pY1221/pY1222) (Cell Signaling, #2249), MET (Cell Signaling, #3127), pMET (Tyr1234/1235) (Cell Signaling, #3077).

For siRNA transfection, ON-TARGETplus single and/or SMARTpool of four siRNA oligos were used to reduce unspecific off-target effects. Control non-targeting and ErbB2, ezrin and radixin targeting oligos were purchased from Thermo Fisher Scientific (Waltham, MA). Cells were transfected with 25 nM siRNA oligos by using Lipofectamine RNAiMAX transfection reagent (Invitrogen Life Technologies, CA) according to the manufacturer's protocol. The cells were transfected for 6 h and subsequently grown in complete growth medium for 3 additional days prior to experiments. For rescue experiments, cells were grown for ~50 h after siRNA transfection. Then they were transfected with 1 μg of plasmid carrying siRNA resistant genes (human ezrin siRNAr sequence; TCCGCAGAACTGAGCTCTG, designed and synthesized by DNA 2.0) per 6 μl FuGENE 6 transfection reagent (Roche) according to the manufacturer's protocol. Control cells were transfected with an empty mammalian expression vector. The cells were then grown for 15 h more prior to experimental procedures.

Total RNA was purified as described previously [17 (link)]. To detect changes in gene expression, we used the following probes, all from Life Technologies: NUMB (Hs01105433_m1), NUMBL (Hs00191080_m1), HES1 (Hs00172878_m1), HES5 (Hs01387463_g1), KLF7 (Hs00748636_s1), NFKB1 (Hs00765730_m1), ERBB2 (Hs01001580_m1), TEAD1 (Hs00173359_m1), TCF4 (Hs00162613_m1), EN1 (Hs00154977_m1), BTRC (Hs00182707_m1), SOX9 (Hs01001343_g1), PTCH1 (Hs00181117_m1), GLI1 (Hs01110766_m1), GLI2 (Hs01119974_m1), CDH1 (Hs01023895_m1), SOX2 (Hs01053049_s1), BMI1 (Hs00995536_m1), NANOG (Hs04260366_g1) and GAPDH (Hs03929097_g1). Quantitative PCR was performed as described previously [17 (link)].

For siRNA transfection as described previously [15 (link)], cells were reversely transfected with RNA iMAX (Life Technologies; 13778–150) and 5 nM scramble siRNA (Life Technologies, 12935–112), kinome siRNA library (2127 siRNA for 709 gene, A30079, Thermo Fisher Scientific), ATG5 (GE Healthcare Dharmacon, 9474), ATG7 (Life Technologies, s20652), BECN1 (Life Technologies, 4392420), ULK1 (Dharmacon, 8408), or ERBB2 (Life Technologies, 4390824;) for 72 h. For shRNA infection, 8XARE reporter plasmid, a packaging and VSV-G expressing envelope plasmid, were transfected into HEK293T cells using the transfection reagent, Lipofectamine 2000 (Life Technologies, 11668–027) for two days to achieve an infection. Cells were then harvested for further experiments or to confirm knockdown efficiency via immunoblotting.