Birc5

Kidneys dissected from embryonic day 14.5 (E14.5) and postnatal day 0 (P0) mice were fixed overnight in 4% paraformaldehyde, embedded in paraffin and sectioned at 4 μm. After deparaffinization, rehydration, and permeabilization in PBS-Tween (PBS-T), antigen retrieval was performed by boiling the slides in 10 mM sodium citrate pH 6.0 buffer for 30 min. Next, sections were blocked in 3% bovine serum albumin (BSA) and incubated overnight with antibodies recognizing BIRC5 (#2808, Cell Signaling Technology, Danvers, MA, USA), Cyclin D1 (#2978, Cell Signaling) and Neural cell adhesion molecule (C9672, Sigma-Aldrich, St. Louis, MO, USA) at the dilutions recommended by the manufacturers. On the next day, sections were washed with PBS-T, incubated with secondary antibodies at the dilution of 1:200, washed again with PBS-T, and mounted in Fluoro Gel with DABCO (Electron Microscopy Science, Hatfield, PA) before being visualized with a Leica DM2500 microscope and photographed with a Leica DFC 7000 T camera using LAS X software (Leica, Buffalo Grove, IL, USA). Goat anti-rabbit 594 (#111–515-047) and donkey anti-mouse 488 (#715–545-151) antibodies were purchased from Jackson InmmunoResearch Laboratories (West Grove, PA, USA).

Protein extraction was performed in protein lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40) supplemented with a cocktail of protease and phosphatase inhibitors (cOmplete Mini, Roche; Phosphatase Inhibitor Cocktail 2 and 3, MilliporeSigma). A total of 30 μg of protein extracts was separated on NuPAGE 4%–12% Bis-Tris Midi Gels (Invitrogen), transferred to a nitrocellulose blotting membrane (GE Healthcare), and blotted with antibodies against the following: EGFR (Abcam, ab52894), phospho-EGFR (Abcam, ab40815), ERK1 (BD Biosciences — Pharmingen, 554100), ERK2 (BD Biosciences, 610103), phospho-ERK1/2 (Cell Signaling Technology, 9101), AKT (Cell Signaling Technology, 9272), phospho-AKT (Cell Signaling Technology, 9271), MEK1 (Santa Cruz Biotechnology Inc., sc-6250), MEK2 (BD Biosciences, 610235), phospho-MEK1/2 (Cell Signaling Technology, 9154), NF-κB p65 (Santa Cruz Biotechnology Inc., sc-372), phospho–NF-κB p65 (Cell Signaling Technology, 3031), STAT3 (Cell Signaling Technology, 9139), phospho-STAT3 (Cell Signaling Technology, 9131), caspase-3 (Cell Signaling Technology, 9662), cleaved caspase-3 (Cell Signaling Technology, 9661), pan-RAS (Calbiochem, OP40), BIRC5 (Cell Signaling Technology, 2808), Lamin B (Santa Cruz Biotechnology Inc., sc-6216), HA.11 (BioLegend, 901513), GAPDH (MilliporeSigma, G8795), and Vinculin (MilliporeSigma, V9131).

Antibodies against the following proteins were used: caspase-3 (#9662), cleaved caspase-3 (#9661), cleaved PARP (#9541), XIAP (#2042), and BIRC5 (#2808) (Cell Signaling Technology, Danvers, MA, USA); β-actin (A5441), LC3B (L7543), and TFAM (SAB1401383) (Sigma, St. Louis, MO, USA); APIP (ab98153), OPA1 (ab42364), and LAMP2 (ab18529) (Abcam, Cambridge, UK); NRF2 (sc-13032) and p62/SQSTM1 (sc-28359) (Santa Cruz Biotechnology, Dallas, TX, USA); and ASCT2 (Proteintech, Rosemont, IL, USA). Gefitinib and erlotinib were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Wako (Tokyo, Japan), respectively. The stable isotope l-glutamine-¹⁵N₂ was purchased from Taiyo Nippon Sanso (Tokyo, Japan).