Nupage 4 12 bis tris midi gel

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).

Proteins were extracted from apical LV samples (n = 12 each group, Day 3) and separated by SDS-PAGE using NuPAGE 4–12% Bis–Tris midi gels (Invitrogen) in criterion cells (Bio Rad) with MOPS SDS running buffer and transferred to nitrocellulose membranes (Biorad) using criterion blotter (Biorad). Membranes were blocked and probed for proteins of interest.

Protein lysates from different brain regions and whole cells were prepared using RIPA buffer with 1X protease inhibitor and 1X phosphatase inhibitor cocktails from Sigma. Denatured protein samples were generated by boiling in 2X Laemmli buffer for 5 min. NuPAGE 4–12% Bis-Tris Midi Gel (Thermo Fisher Scientific) was used for electrophoresis. To enable the comparison of PERK knockdown efficiency in different brain regions, protein quantification was performed on protein lysates from brain tissue using Peirce BCA protein assay kit (Thermo scientific, # 23227), and 50μg protein per sample was loaded for western blot. The following primary antibodies were used in western blot analysis: monoclonal anti-PERK produced in rabbit (1:500, cell signaling, #3192), monoclonal anti-β-actin produced in mouse (1:1000, GenScript, A00702), monoclonal anti-p-PERK produced in rabbit (1:500, cell signaling, #3179), polyclonal anti-eIF2α [pS⁵²] produced in rabbit (1:1000, Invitrogen, 44728G), monoclonal anti-α-tubulin produced in mouse (1:1000, Sigma, T5168).

40 μg of brain extracts were loaded onto a NuPAGE 4–12% Bis–Tris Midi Gel (Thermo Fisher Scientific, Cat# WG1402BOX) and electrophoresed at 130 V for 2 hr with NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, Cat# NP0001-02). Proteins were then electrophoretically transferred onto a nitrocellulose membrane (GE Healthcare, Amersham Protran Supported 0.45 µm NC) at 100 V for 90 min on ice in transfer buffer (48 mM Tris–HCl and 39 mM glycine supplemented with 20% (v/v) methanol). The transferred membrane was blocked with 5% (w/v) skim milk powder dissolved in TBS-T (50 mM Tris base, 150 mM sodium chloride (NaCl), 0.1% (v/v) Tween 20) at room temperature (RT) for 30 min and incubated overnight at 4°C in primary antibodies diluted in 5% (w/v) BSA in TBS-T. After incubation with primary antibodies, membranes were washed three times for 5 min with TBS-T and incubated with near-infrared fluorescent dye-labelled secondary antibodies (diluted to 1:20,000) for 1 hr at RT. Thereafter, membranes were extensively washed with TBS-T and protein bands were acquired via near-infrared fluorescent detection using the Odyssey CLx imaging system and the signal intensity quantified using Image Studio software.

Proteins in LDS-loading buffer were separated on a NuPAGE 4–12% Bis–Tris Midi Gel (WG1403A, Thermo Fisher) or on a Bolt™ 4–12% Bis–Tris Plus Gel (NW04120BOX, Thermo Fisher). The gel was incubated for 2 h at room temperature in fixing solution (50% MeOH, 12% HAc, 0.05% formalin). The gel was subsequently washed 3 × 20 min at room temperature in 35% EtOH. Thereafter, the gel was sensitized in 0.02% Na₂S₂O₃ for 2 min, washed 3 × 5 min in ultrapure water and incubated for 20 min at room temperature in silver staining solution (0.2% AgNO₃, 0.076% formalin). Then, the gel was washed 2 × 1 min in ultrapure water and developed in developing solution (6% Na₂CO₃, 0.05% formalin, 0.0004% Na₂S₂O₃). Upon desired band intensities, the reaction was stopped by replacing developing solution with stop solution (50% MeOH, 12% HAc) and incubating for 5 min.