Rabbit anti gfp

Cells grown on coverslips were washed with PBS. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 20 min. Cells were rinsed with PBS+ (0.1% saponin (Sigma-Aldrich) and 0.02% NaN₃ (Sigma-Aldrich) in PBS, pH 7.4) and blocked with PBS+BSA (20 mg/mL bovine serum albumin (BSA; Sigma-Aldrich) in PBS+) for 30 min. Cells were then incubated overnight at 4°C with mouse anti-Hb9 (1∶10; clone 81.5C10, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), mouse anti-Tuj1 (1∶1000; Covance, Princeton, NJ, USA), mouse anti-Islet-1 (1∶50; clone 40.2D6, DSHB), mouse anti-nestin (1∶500, Millipore), or mouse anti-NeuN (1∶200, Millipore) in PBS+BSA supplemented with rabbit anti-GFP (1∶2000, Rockland Immunochemicals, Gilbertsville, PA, USA). After three washes with PBS+, cells were incubated for 1 hr at room temperature with Alexa Fluor 594 goat anti-mouse IgG (Life Technologies) and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) in PBS+BSA. Cells were washed three times with PBS+ and incubated with 100 ng/mL Hoechst 33342 (Life Technologies) in ddH₂O for 10 min and rinsed with ddH₂O before mounting in Immu-Mount (Shandon-Lipshaw). Images were obtained using a Leica TCS SP5 confocal microscope.

Primary antibodies used were rabbit anti-GFP (Rockland, 600-401-215, 1:200) and mouse anti-EGFR (Santa Cruz, sc120, 1:200). Secondary antibodies used were goat anti-mouse Alexa Fluor 546 (Invitrogen, A11030, 1:500), donkey anti-rabbit Alexa Fluor 594 (Invitrogen, A21207, 1:500), goat anti-mouse quantum dot (QD) 655 (Life Technologies, Q11021MP, 1:500), goat anti-rabbit QD655 (Life technologies, Q11421MP, 1:500), sheep anti-mouse HRP (GE-Healthcare, NA931V, 1:500), donkey anti-rabbit HRP (GE-Healthcare, NA934V, 1:500), goat anti-mouse 10 nm gold (BBI solutions, EM.GMHL10, 1:50), and goat anti-rabbit 10 nm gold (BBI solutions, EM.GAR10, 1:50).

To prepare whole‐cell extracts, cells (5 × 10⁵) were harvested and resuspended in 250 μL of 1× NuPAGE LDS sample buffer (NP0008; Thermo Fisher Scientific K.K., Yokohama, Japan). Whole‐cell extracts were sonicated briefly to reduce viscosity, and 10 μL of the extract was subjected to electrophoresis on 4–12% Bis–Tris NuPAGE gels (NP0321; Thermo Fisher Scientific K.K.). Proteins were transferred to polyvinylidene fluoride membranes and probed using rabbit anti‐GFP (600‐401‐215; Rockland Immunochemicals, Pottstown, PA, USA), anti‐p62/SQSTMI (P0067; Sigma‐Aldrich), or anti‐glyceraldehyde 3‐phosphate dehydrogenase (anti‐GAPDH) antibody (14C10; Cell Signaling Technology, Danvers, MA, USA) and secondary antibodies conjugated to horseradish peroxidase (NA9340; GE Healthcare Life Sciences). Protein bands were stained with ImmunoStar Zeta (295‐72404; Wako Pure Chemical Industries) and detected by chemiluminescence using a ChemiDoc MP imaging system (Bio‐Rad, Tokyo, Japan).