Journal: Frontiers in Veterinary Science

Article Title: Effect of Cyclic Heat Stress on Hypothalamic Oxygen Homeostasis and Inflammatory State in the Jungle Fowl and Three Broiler-Based Research Lines

doi: 10.3389/fvets.2022.905225

Figure Lengend Snippet: Effect of HS on hypothalamic expression of AMPK pathway in three modern broilers and their ancestor JF. Protein and RNA were extracted from hypothalamic tissues and analyzed by western blot (A,B) and qPCR (C–I) , respectively. Gene expression was determined by qPCR using 2 −ΔΔCT method and data are mean ± SEM ( n = 6/group). Protein expression was presented as ratio of p-AMPK Thr172 /pan AMPK (A,B) . Protein was analyzed via AlphaVIew software and is expressed as mean ± SEM ( n = 3/group) with one representative blot shown (A) . Different letters indicate significant difference at P < 0.05. 95RAN, 1995 random bred; ACRB, Athens Canadian Random Bred; AMPK, AMP-activated protein kinase; E, environment; ExL, interaction between E and L; HS, heat stress (36°C); JF, jungle fowl; L, line; MRB, modern random bred; TN, thermoneutral (25°C).

Article Snippet: The primary antibodies used were rabbit polyclonal anti-phospho AMPKα1/2 Thr172 (#2531, Cell Signaling Technology, Danvers, MA), anti-AMPKα1/2 (#2795, Cell Signaling Technology, Danvers, MA), anti-HSP90 (#PA5-17610, Pierce Thermo Scientific, Rockford, IL), goat polyclonal anti-HSP60 (#sc-1052, Santa Cruz Biotechnology, Dallas, TX), mouse monoclonal anti-HSP70 (#MAI-91159, Pierce Thermo Scientific, Rockford, IL) and rabbit anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Expressing, Western Blot, Gene Expression, Software

Journal: bioRxiv

Article Title: Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of cerebellar CHIPopathy

doi: 10.1101/283192

Figure Lengend Snippet: ( A ) Immunoblot analysis of CHIP and β-tubulin protein or ( B ) qPCR analysis of Stub1 mRNA levels in primary fibroblasts isolated from T246/T246 (T/T), T246/M246 (T/M), or M246/M246 (M/M) mouse embryos. Relative mRNA levels are represented by the dot plot and summarized by the mean and 95% confidence intervals. ( C ) Immunoblot analysis of CHIP and β-actin protein in fibroblasts isolated from control patients (WT) or siblings that are homozygous for CHIP-T246M (II-1 and II-2). ( D ) Immunoblot analysis of CHIP and β-tubulin in soluble cell lysates from either T/T or M/M, fibroblasts, or fibroblasts isolated from CHIP-/-embryos (KO) after exposure to cycloheximide (CHX) indicated in hours (h). Two exposures of CHIP immunoblots are provided to help visualize M/M conditions. ( E ) Immunoblot analysis of CHIP and β-actin in either the soluble or insoluble fraction of lysates or whole cell lysates from T/T or M/M MEFs treated with 20 µM MG132 or 0.05% DMSO control for 4 hours. ( F ) Immunoblot analysis of AMPKα1 and CHIP in cell lysates from T/T or M/M MEFs either before (input) or after immunoprecipitation (IP) of the indicated protein. Control samples (C) contained a mixture of 50% T/T and T/M and were immunoprecipitated with either rabbit IgG or goat IgG as controls for the CHIP and AMPKα1 antibodies, respectively. ( G ) Immunoblot analysis of HSC70 and CHIP in cell lysates from T/T or M/M MEFs either before (input) or after immunoprecipitation (IP) of CHIP. Control samples (C) contained a mixture of 50% T/T and T/M and were immunoprecipitated with rabbit IgG to control for the CHIP antibody. ( H ) Immunoblot analysis of HSP70 and CHIP in MEFs with the indicated genotypes that were treated without heat shock (no) or with heat shock (HS) followed by the indicated recovery time.

Article Snippet: Beads were washed four times with Phosphate-Buffered Saline with 0.05% Tween-20; subsequently, proteins were eluted in SDS-sample buffer and analyzed by SDS-PAGE and western blotting using anti-CHIP (Sigma, S1073), anti-AMPKα1/2 (Cell Signaling Technologies, 2532) or anti-Hsc70 (Enzo, ADI-SPA 815) antibodies.

Techniques: Western Blot, Isolation, Immunoprecipitation