Sc 200

Total cellular and nuclear protein was extracted from retinas and quantified using bicinchoninic acid assay kit. Homogenate in 2 × sodium dodecyl sulfate (SDS) sample buffer was boiled for 5 min, and then equal amounts of protein (40 μg) from each sample were subjected to electrophoresis on a 10% (v/v) SDS-polyacrylamide gel. After proteins were electroblotted to a polyvinylidene difluoride membrane, the membrane was blocked with Phosphate-buffered saline containing 5% dried non-fat milk or 3% BSA at room temperature for 1 h, and incubated with indicated primary antibodies (anti-ATF4, 1:1000, SC-200, Santa Cruz Biotechnology, Dallas, TX; anti-ATF6, 1:1500, ab37149; anti-GRP78/BiP, 1:2000, ab21685; anti-pIRE1α, 1:1000, ab48187, Abcam, Cambrigde, MA; anti-Nrf2, 1:1000, SC-722; Santa Cruz Biotechnology, Dallas, TX; anti-p38, 1:500, ab27986; anti-p-p38, 1:1000, ab4822; anti-JNK, 1:1000, ab59227; anti-p-JNK, 1:2000, ab124956; anti-p65, 1:600, ab7970; anti-p-p65, 1:2000, ab86299; anti-SIRT1, 1:2000, ab12193, Abcam, Cambrigde, MA) at 4 °C overnight, followed by incubating with the goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody for 2 h. After incubation, membrane was washed three times, and the antigen-antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer, Akron, OH).

Differential centrifugation was performed as described above for 6xHis-3xFLAG-ATF6a expressing T-Rex cells except the buffer volumes were quartered. For western blot analysis, 10 μL of total or 15 μg nuclear extract was loaded per lane of a fifteen well SDS-PAGE gels, one gel each for ATF6α and ATF6β. After blotting, membranes were probed with antibodies against ATF6α (Haze et al., 1999 (link)) (1:1000 in 5% BSA) or ATF6β (Wu et al., 2007 (link)) (1:1000 in 5% milk) at four degrees overnight. After developing for ATF6α and ATF6β membranes were stripped for sixty seconds shaking at room temperature in stripping buffer (7 M Guanidine hydrochloride (Sigma G4505), 50 mM Glycerol (MP Biomedicals 800689), 50 μM EDTA (Fisher BP120-1), 100 μM potassium chloride (Fisher P217-3), 20 mM β-mercaptoethanol (Sigma M6250), washed twice in distilled water, stripped again for sixty seconds, washed extensively with distilled water, PBS-Tween, blocked in 5% milk before cutting the membranes to probe for PERK (1:500, Cell Signaling Technology C33E10) or ATF4 (1:1000, Santa Cruz sc-200) each in 5% milk in PBS-Tween.

Commercially available primary antibodies against the following antigens were used: eIF2α phosphorylated at Ser51: rabbit monoclonal, 119A11 (Cell Signaling) and rabbit monoclonal, ab32157 (Abcam); total eIF2α: rabbit monoclonal, D7D3 (Cell Signaling); LC3B: rabbit polyclonal, L7543 (Sigma); HSP70: mouse monoclonal, C92F3A-5 (Enzo Life Sciences); GFP: mouse monoclonal (sc-9996, Santa Cruz); ATF4: rabbit polyclonal, sc-200 (Santa Cruz); CHOP: mouse monoclonal, clone L63F7 (Cell Signaling); TIAR: mouse monoclonal, 610352 (BD Biosciences); cleaved caspase 3: rabbit polyclonal, 9661S (Cell Signaling); puromycin: mouse monoclonal, clone 12D10 (Merck Millipore); beta-actin: mouse monoclonal, A5441 (Sigma). Antibodies were used at 1:1000 dilution for all applications.

Methods used were as described previously (Tsang et al., 2007 (link)). In brief, limbs were fixed in 4% PFA, followed by demineralization in 0.5M EDTA (pH 8.0) before embedding in paraffin. Slides were stained with Alcian Blue for cartilage matrix and Fast Red for nuclei. Immunofluorescence was performed using antibodies against ATF4 (sc-200, Santa Cruz), ATF3 (HPA001562, Sigma), CHOP (sc-575, Santa Cruz), PPP1R15A (sc-825, Santa Cruz), FGF21 (42189, AIS) and Sox9 (AB5535, Millipore).

Murine recombinant insulin-like growth factor-1 (IGF-1) was purchased from PeproTech (Rocky Hill, NJ). Thapsigargin was obtained from Applichem (St Louis, MO), tunicamycin from Sigma (St Louis, MO) and brefeldin A from TOKU-E (Bellingham, WA). mTOR and PERK inhibitors were purchased from LC Labs (Woburn, MA) (rapamycin), Selleckchem (Houston, TX) (WYE125132, GSK2656157), Abcam (Cambridge, UK) (PP242) and from GlaskoSmith and Kline (Middlesex, UK) (GSK2126458). Antibodies against phospho-PERK (Thr980) (No.3179), PERK (No. 3192), phospho-eIF2α (Ser51) (No. 3597), eIF2α (No.2103), phospho-p70S6K1^thr389 (No. 9234), p70S6K1 (No. 9202) and cleaved-caspase3 (No. 9664) were purchased from Cell Signaling Technology (Beverly, MA). Phospho-4E-BP1 (phosphorylation on Thr45; ab68187), and 4E-BP1 (ab2606) were from Abcam (Cambridge, UK). ATF6 clone 70B1413.1 were from Abcam (Cambridge, UK; ab11909) and Novus Biological (Littleton, CO; NBP1-40256). Antibodies against CHOP (SC-575), ATF4 (SC-200), GADD34 (SC-8327) and XBP1 (SC-7160) came from SantaCruz Biotechnology (Santa Cruz, CA) and BiP (610978) from BD Laboratories^™ (Franklin Lakes, NJ). Antibody against α-tubulin was from Sigma-Aldrich (St. Louis, MO).