Perk1 2 antibodies

TMA slides were deparaffinized and rehydrated according to common protocols. Antigen retrieval was performed by boiling the slides in citrate buffer (pH = 6.0) for 10 min using a microwave oven. Sections were blocked using solution of 10% FBS (Thermo Fisher Scientific) and 1% BSA in TBS for 2 h at room temperature and probed with pERK1/2 antibodies (Cell Signaling Technology, D13.14.4E, 1:100 in TBS with 1% BSA) overnight at 4 °C. Slides were rinsed with 0.025% Triton-X100 (Fisher Scientific) in TBS, probed with secondary HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, 7074, 1:1000 in TBS with 1% BSA) for 1 h at room temperature and developed with a 3,3-diaminobenzidine kit (Vector Laboratories, Burlingame, CA, USA). Separate slides were counterstained with hematoxylin (Fisher Scientific). After staining slides were rinsed with DI water, dehydrated and mounted using toluene (Fisher Scientific).
Tissue slides with tumor samples obtained from mice injected with PEO4 cells (see below) were stained at the histology core at the University of Michigan using EDTA-based antigen retrieval and mouse anti-ALDH antibody (BD Biosciences, San Jose, CA, USA, clone 44/ALDH; 1:100) as previously described [43 (link)]. For stain quantification, five sections from three tumors per treatment group were analyzed by two people. Counts were then compared using a 2-sided Student’s t test.

Tissue slides were deparaffinized and rehydrated according to common protocols. Antigen retrieval was performed by boiling the slides in citrate buffer (pH = 6.0) for 10 min using a microwave oven. Sections were blocked using solution of 10% FBS (Thermo Fisher Scientific) and 1% BSA in TBS for 2 h at room temperature and probed with pERK1/2 antibodies (Cell Signaling Technology, D13.14.4E, 1:100 in TBS with 1% BSA) overnight at 4 °C. Slides were rinsed with 0.025% Triton-X100 (Fisher Scientific) in TBS, probed with secondary Alexa488-conjugated anti-rabbit IgG (Cell Signaling Technology, 4412, 1:1000 in TBS with 1% BSA) for 1 h at room temperature and counterstained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Millipore Sigma). After staining slides were rinsed three times with TBS, coverslips were mounted using PermaFluor medium (Thermo Fisher Scientific).

SAHA (Purity ≥99%) was purchased from Selleck Chemicals (Houston, TX). Matrigel and the anti-Semaphorin-4D (Sema-4D) antibody were obtained from BD Biosciences (San Jose, CA). Trypan blue was purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC apoptosis detection kit was purchased from Biotech Co., Ltd (Nanjing, China). RNase-free DNase I was from Qiagen (Hilder, Germany). RevertAid™ First Strand cDNA Synthesis Kit was purchased from Fermentas Life Sciences (Chicago, IL). Taq™ DNA Polymerase was from TaKaRa Biotechnology Co., Ltd (Dalian, China). Propidium iodide (PI), monoclonal antibody against β-actin and gelatin were obtained from Sigma (St. Louis, Mo). The anti-cyclin-D1 antibody was obtained from ABGENT (Suzhou, China). Anti-epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). Akt, p-Akt (Ser 473), p70S6 kinase (S6K1), p-S6K1 (Thr 389), S6, p-S6 (Ser 235/236), mTOR, p-mTOR (Thr 289), Ulk1, p-Gsk-3β, Ulk1, Erk1/2 and p-Erk1/2 antibodies were purchased from Cell Signaling Tech (Beverly, MA). Primers were synthesized by GENEWIZ, Inc. (Suzhou, China).