Anti p perk

Forty-eight hours post transfection cellular lysates or cellular medium were collected for experiments. Cells were lysed in equal volumes of pre-heated 2xSDS loading buffer (Sodium Dodecyl Sulphate 125 mM, Tris-HCl pH 6.8, 20% Glycerol, 2% SDS, 2% β-mercaptoethanol and bromophenol blue) and homogenized via sonication. Antibodies used against the ER-Stress proteins were: anti-BiP, anti-PERK, anti-P-PERK (Cell Signaling Technology, Danvers, MA) and anti-CHOP, anti-p-eIF2a (SantaCruz Biotechnology, CA), followed by peroxidase-labelled secondary antibodies either goat anti-mouse or donkey anti-rabbit (SantaCruz Biotechnology, CA). Proteins were detected using the Enhanced ChemiLuminescence (ECL) Plus Blotting Detection system (Amersham Biosciences, Buckinghamshire, UK) and were visualized by autoradiography on photographic film (KODAK X-OMAT, NY). All transblots were reprobed with anti-β-tubulin antibody (SantaCruz Biotechnology, CA) to prove equal amounts of protein were loaded on the membrane. Band density was defined using the ImageJ Software (http://imagej.nih.gov/ij).

Study of human marrow specimens was approved by the Institutional Research Board (IRB) of the University Hospitals Case Medical Center. The study includes 5non-leukemia control and 4 T-ALL patient BM sections. Expression of p-PERK, p-eIF2a and JAG1 were evaluated by immunohistochemistry (IHC). Sections were incubated with the primary antibodies overnight at 4°C (anti p-PERK, 1:200, Cell signaling; anti-p-eIF2a,1:50, Cell Signaling; anti-JAG1, 1:1000, Sigma, St. Louis, MO). Images were analyzed on an Aperio Image Scope.

The following antibodies were used in this study: anti-USP33, anti-slug, and anti-twist1 antibodies were purchased from ProteinTech Group (Chicago, IL, USA); anti-CXCR4 antibody was purchased from BD Bioscience (Franklin Lakes, NJ, USA); anti-HA and anti-Flag M1 antibodies were from Sigma-Aldrich (St. Louis, MO, United States); anti-ppERK, anti-ERK, and anti-β-arrestin2 antibodies were from Cell Signaling Technology (Boston, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-snai1, and anti-β-actin antibodies were purchased from Santa Cruz (Dallas, TX, USA).
N-ethylmaleimide (NEM, deubiquitinase inhibitor), SDF-1 (SDF-1α, CXCR4 agonist), AMD3100 octahydrochloride hydrate (1,1′-[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride, CXCR4 antagonist), dynasore hydrate (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide hydrate, dynamin inhibitor), HA affinity beads, and Flag affinity beads were all purchased from Sigma-Aldrich.

Cell or tissue lysates were extracted in RIPA buffer (Beyotime, Cat# P0013B) Protein concentration was evaluated by bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Cat# 23227) and then analyzed by SDS–PAGE gel electrophoresis and blotting onto PVDF membranes. Primary antibodies used included: anti-ERO1L (Abcam, Cat# ab177156), anti-IRE1α (Abcam, Cat# ab37073), anti-pIRE1α (Abcam, Cat# ab48187), anti-XBP1s (Cell Signaling Technology, Cat# 12782), anti-PERK (Abcam, Cat# ab229912), anti-pPERK (Cell Signaling Technology, Cat# 3179), anti-eIF2α (Cell Signaling Technology, Cat# 9722), anti-peIF2α (Cell Signaling Technology, Cat# 9721), anti-ATF4 (Cell Signaling Technology, Cat# 11815), anti-ATF6α (Santa Cruz, Cat# sc-166659), anti-CHOP (Santa Cruz, Cat# sc-7351), and anti-β-Actin. Primary antibodies were applied in 5% non-fatty milk or primary antibody dilution (Beyotime, Cat# P0256) in TBST and incubated overnight at 4°C, followed by HRP-conjugated secondary antibodies incubation at room temperature for 2 h. Secondary antibodies used included: Goat anti-Rabbit HRP-conjugated IgG (Abcam, Cat# ab6721), Rabbit anti-Mouse HRP-conjugated IgG (Abcam, Cat# ab6728). Images were collected by Amersham Imager 600 (General Electric).