Journal: PLoS ONE

Article Title: Masseter Muscle Myofibrillar Protein Synthesis and Degradation in an Experimental Critical Illness Myopathy Model

doi: 10.1371/journal.pone.0092622

Figure Lengend Snippet: Western blot analyses of A ) MuRF1, B ) atrogin-1, C ) LC3b-I (black), LC3b-II (grey), D ) Akt (black) and phosphorylated Akt (grey), E ) MMP-2, F ) TIMP-2, G ) HSP70, H ) αB-crystallin, I ) HSP90. Values are normalized to α-actin content in the masseter muscle in controls and rats exposed to immobilization, mechanical ventilation, post-synaptic neuromuscular blockade for 0.25–4, 5–8 and 9–14 days. N = 4 per group. Asterisk (*p<0.05, **p<0.01) denotes significant difference compared with controls, and hash # p<0.05 denotes significant difference compared with 0.25–4 day group (MMP-2) or 9–14 day group (p-Akt). Values are means of optical intensity (arbitrary units) + SEM.

Article Snippet: Membranes were incubated with primary antibodies of MuRF1 (AF5366, R&D Systems, MN, USA), atrogin-1 (AP2041 ECM Biosciences, KY, USA), HSP70, HSP90 and αB-crystallin (SMC-100B, SMC-137C and SMC-159A, respectively, StressMarq Biosciences Inc. Canada), LC3b (L754, Sigma-Aldrich, Munich, Germany), Akt and p-AKt (9272, 9271 respectively, Cell Signaling Technology, Inc., Danvers, Canada), MMP-2 and TIMP-2 (MAB3308, CC1064, respectively, Merck Millipore, USA), and actin (sc-1616-R, Santa Cruz Biotechnology Inc., CA, USA).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels *

doi: 10.1074/jbc.M116.752618

Figure Lengend Snippet: Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting Hsp70 interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.

Article Snippet: The following commercially available antibodies were used: Bag1 (Santa Cruz Biotechnology); CHIP (Abcam); goat anti-rabbit IgG-conjugated HRP (Jackson ImmunoResearch); goat anti-mouse IgG-conjugated HRP (Sigma); gp78 (Abcam); hemagglutinin (HA.11) (Covance); hERG (Alomone Labs); HRD1 (Abcam); Hsc70 (StressMarq); Hsp70 (StressMarq); Hsc/Hsp70 (StressGen); RMA1 (Abcam);TRC8 (Abcam); tubulin (Sigma); Ube2j1 (Abcam); Ube2g2 (Abcam); and ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Construct, Mutagenesis, Stable Transfection, Western Blot, Plasmid Preparation, Quantitation Assay, Fluorescence, Microscopy, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels *

doi: 10.1074/jbc.M116.752618

Figure Lengend Snippet: Bag1 disrupts chaperone complexes with hERG. A, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control, and hERG was immunoprecipitated (IP). Co-precipitating Hsc70/Hsp70 and CHIP were detected by immunoblot (IB); Hsc70 and Hsp70 were equally detected by the antibody used here. Quantitation of Hsc70/Hsp70 and CHIP was adjusted for the total amount of immunoprecipitated hERG and plotted relative to the amounts in the control. Averages and standard deviations are shown to the right of data points. B, HeLa cells stably expressing hERG were transfected with siRNA against both Hsc70 and Hsp70, or against CHIP, or non-silencing control. CG and FG hERG were detected by immunoblot and quantified relative to the amount of each in control cells. C, cells as in B transfected with siRNA against Hsc70/Hsp70 were examined for hERG kinetics by pulse-chase as in Fig. 2A. Data points and lines representing the averages are shown. D, HEK293 cells were transfected with hERG and either Bag1 or vector control. Total light membrane fractions were isolated and treated with the indicated amounts of trypsin for 10 min at 37 °C. CG and FG hERG were detected by immunoblot and quantified relative to the amount without trypsin treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to controls.

Article Snippet: The following commercially available antibodies were used: Bag1 (Santa Cruz Biotechnology); CHIP (Abcam); goat anti-rabbit IgG-conjugated HRP (Jackson ImmunoResearch); goat anti-mouse IgG-conjugated HRP (Sigma); gp78 (Abcam); hemagglutinin (HA.11) (Covance); hERG (Alomone Labs); HRD1 (Abcam); Hsc70 (StressMarq); Hsp70 (StressMarq); Hsc/Hsp70 (StressGen); RMA1 (Abcam);TRC8 (Abcam); tubulin (Sigma); Ube2j1 (Abcam); Ube2g2 (Abcam); and ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Quantitation Assay, Stable Transfection, Expressing, Pulse Chase, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels *

doi: 10.1074/jbc.M116.752618

Figure Lengend Snippet: TRC8 decreases trafficking and function of hERG. A, HEK293 cells were transfected with hERG and the indicated TRC8 construct or vector control and examined for hERG kinetics by pulse-chase as in Fig. 2A. Data points and lines representing the averages are shown. B, HEK293 cells were transfected with hERG, GFP, and either TRC8 or vector control. Voltage-response curves from patch clamp measurements as in Fig. 1E are shown. C, cells were transfected as in A with the indicated HA-tagged TRC8 construct, and hERG was immunoprecipitated (IP). Co-precipitating TRC8 was detected by immunoblot using specific antibodies against TRC8 and the HA tag, and Hsp70 was detected as a control. Quantitation of TRC8 was adjusted for the total amount of immunoprecipitated hERG and plotted relative to the amounts in the control. *, p < 0.05; **, p < 0.01 relative to controls.

Article Snippet: The following commercially available antibodies were used: Bag1 (Santa Cruz Biotechnology); CHIP (Abcam); goat anti-rabbit IgG-conjugated HRP (Jackson ImmunoResearch); goat anti-mouse IgG-conjugated HRP (Sigma); gp78 (Abcam); hemagglutinin (HA.11) (Covance); hERG (Alomone Labs); HRD1 (Abcam); Hsc70 (StressMarq); Hsp70 (StressMarq); Hsc/Hsp70 (StressGen); RMA1 (Abcam);TRC8 (Abcam); tubulin (Sigma); Ube2j1 (Abcam); Ube2g2 (Abcam); and ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Plasmid Preparation, Pulse Chase, Patch Clamp, Immunoprecipitation, Western Blot, Quantitation Assay