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

Journal: PLoS Biology

Article Title: Composition, Formation, and Regulation of the Cytosolic C-ring, a Dynamic Component of the Type III Secretion Injectisome

doi: 10.1371/journal.pbio.1002039

Figure Lengend Snippet: Strains expressing YscQ M218A from the native promoter and therefore lacking YscQ C do not secrete effectors and YscQ M218A requires YscQ C for localization at the injectisome. YscQ C and YscQ M218A colocalize; increased expression levels of mCherry-YscQ C lead to an increase in spot number, but not spot intensity for EGFP-YscQ M218A . (A) Secretion assay showing the secreted proteins in a wild-type (WT) strain, and YscQ M218A , uncomplemented or complemented in trans with YscQ C , EGFP-YscQ C , or mCherry-YscQ C . (B) Fluorescence micrographs showing the distribution of EGFP-YscQ, EGFP-YscQ C , and EGFP-YscQ M218A , uncomplemented or complemented by YscQ C in trans . (C) Cellular distribution of EGFP-YscQ M218A (expressed from its native promoter on the virulence plasmid, second row) and mCherry-YscQ C (expressed in increasing amounts in trans induced by the given concentrations of arabinose, third row). The overlay (bottom row) displays the colocalization of both YscQ versions. Scale bars, 2 μm. (D) Number of detected EGFP-YscQ M218A foci per bacterium in cells expressing increasing amounts of mCherry-YscQ C (as in (C)). n > 170 cells per condition (see for details). Black lines represent the average number of foci per bacterium; circles represent the number of foci per single bacterium (arranged in groups of ten). ***, p < 0.001. (E) Average number of detected EGFP-YscQ M218A foci per bacterium in relation to the amount of mCherry-YscQ C , as quantified on an immunoblot with a polyclonal anti-YscQ antibody . Data points from left to right: no YscQ C (no plasmid), and increasing amounts of mCherry-YscQ C (no arabinose, 0.001%, 0.002%, 0.004%, 0.01%, 0.02% arabinose, as in (C)). (F) Average intensity of foci for the bacteria analyzed in (D)). Error bars represent standard deviations of all foci. Numerical values and raw data for (D–F) can be found in .

Article Snippet: Immunoblotting was carried out using mouse polyclonal antibodies against GFP (Clontech 632459, 1:1,000) or mCherry (Clontech 632543, 1:1,000) or rabbit polyclonal antibodies against YscQ (MIPA235, 1:1,000).

Techniques: Expressing, Fluorescence, Plasmid Preparation, Western Blot

Journal: International Journal of Neuropsychopharmacology

Article Title: Plasticity of Functional MAOA Gene Methylation in Acrophobia

doi: 10.1093/ijnp/pyy050

Figure Lengend Snippet: Functional analysis of MAOA promoter/exon I/intron I DNA methylation using luciferase-based reporter gene assays. Left: Normalized reporter gene activity was significantly decreased in the presence of pCpGfree-promoter Lucia_MAOA vectors containing the methylated insert spanning CpGs 1–13 compared with those carrying a nonmethylated insert; *** P < .001. Right: No significant difference in normalized reporter gene activity was discerned between methylated or non-methylated pCpGfree-promoter Lucia control vectors lacking the insert of the sequence spanning CpGs 1–13.

Article Snippet: Functional analysis was accomplished using the pCpGfree-promoter- Lucia vector (InvivoGen) expressing a Lucia luciferase under a human elongation factor-1 (hEF1) promoter.

Techniques: Functional Assay, DNA Methylation Assay, Luciferase, Activity Assay, Methylation, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *

doi: 10.1074/jbc.M113.546283

Figure Lengend Snippet: Effect of DNA methylation on NPC1L1 promoter activity. Human NPC1L1 promoter region flanking the area between −1741 and +56 (+1 is the transcription initiation site) was amplified by PCR and cloned into a promoterless CpG free vector (pCpG free basic lucia) upstream of the lucia gene. The NPC1L1 promoter construct as well as the empty vector were then subjected to in vitro methylation. The methylated and unmethylated empty vector (pEV) and NPC1L1 promoter construct (L1p) were transiently transfected into Caco2 cells along with the mammalian expression vector of β-galactosidase. 48 h post-transfection, cells were harvested, and lucia activity and β-galactosidase activity were measured. The relative promoter activity was determined as a ratio of lucia to β-galactosidase (A). Data are presented as fold increase compared with unmethylated empty vector and represent mean ± S.E. of three independent experiments performed on separate occasions. *, p < 0.05 compared with empty vector pEV. CpG free EF1 promoter lucia vector (pCpG free lucia) was used as control for methylation as described under “Results” (B). Data are presented as fold change relative to unmethylated vector.

Article Snippet: For the construction of pCpG free L1 vector, the DNA fragment containing −1741/+56 region of the human NPC1L1 promoter was amplified using forward 5-ATCGATGCGAGTACTTGGACTCTATCTCTCTGTGG-3′ and reverse 5-ATCGATGCGAAGCTTCCCAGGTCTGGGAAGGGGTCA-3′ primers, and the amplified promoter fragments were then inserted into a promoterless CpG free vector (pCpG free basic lucia, InvivoGen, San Diego) upstream of the lucia reporter gene.

Techniques: DNA Methylation Assay, Activity Assay, Amplification, Clone Assay, Plasmid Preparation, Construct, In Vitro, Methylation, Transfection, Expressing, Control

Journal: Cerebellum (London, England)

Article Title: Functional convergence of motor and social processes in lobule IV/V of the mouse cerebellum

doi: 10.1007/s12311-021-01246-7

Figure Lengend Snippet: (a) Mice were randomly divided into AAV5-hSyn-EGFP (control), NMDA (lesion) and AAV8-Pcp2-hM3Dq-mCherry (PC-M3) groups, followed by a series of behavioral tests. Clozapine-N-oxide (CNO, 1 mg/kg) was given to all the groups before each test. Mice were sacrificed 90 min after the final test (free social interaction, FSI) and c-Fos immunostaining was performed. (b) Examples of the sagittal view of cerebellar slices from the control, lesion and PC-M3 groups. Fluorescence in lobule IV/V was EGFP (control), DAPI (lesion) or mCherry (PC-M3). Zoom-in images are shown in bottom panels. In the control group, EGFP was found preferentially in granule cells and interneurons. In the lesion group, cell loss was seen in molecular layer (ML), Purkinje cell (PC) layer, and granular layer (GL). In the PC-M3 group, only PCs were labelled with mCherry. (c) Diagrams of the coronal view of cerebellar sections. Affected areas in lobule IV/V are highlighted in green (EGFP in control group), grid-shaped (lesions in lesion group) and red (mCherry in PC-M3 group), respectively. Numbers indicate distance relative to the bregma.

Article Snippet: Minimal Pcp2/L7 promoter was inserted into pAAV-CaMKIIa-hM3D(Gq)-mCherry (Addgene #50476, a gift from Dr. Bryan Roth) to confer specific expression of a human M3 muscarinic receptor (hM3Dq) to PCs (PC-M3) [ 31 , 37 ].

Techniques: Immunostaining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Hsp70 and Hsp90 Multichaperone Complexes Sequentially Regulate Thiazide-sensitive Cotransporter Endoplasmic Reticulum-associated Degradation and Biogenesis *

doi: 10.1074/jbc.M113.455394

Figure Lengend Snippet: The TPR cochaperone/E3 ubiquitin ligase CHIP stimulates the chaperone-dependent ERAD of NCC. A, domain architecture of CHIP and the ΔUbox and ΔTPR deletion mutants used for these studies. B, analysis of steady state HA-NCC expression by immunoblotting in HEK293T cells coexpressing either full-length Myc-tagged CHIP, the ΔUbox Myc-CHIP mutant, or empty vector (pcDNA3.1). Mature and core NCC glycoforms are indicated with arrowheads. Parallel immunoblots of Hsc70 were performed as a loading control. C, cycloheximide chase analysis of NCC degradation. MDCK cells transiently expressing HA-NCC with either empty pcDNA3.1 vector or Myc-CHIP were treated for 3 h with 100 μg/ml cycloheximide as described under “Experimental Procedures.” At the indicated time points, cells were lysed, and whole cell lysates were subsequently probed for HA-NCC using anti-HA antibodies. Top panel, quantification of core-glycosylated NCC degradation in the absence and presence of CHIP. *, p < 0.012 by Student's t test (n = 3). Representative immunoblots for both experimental groups are shown below the graph. D, effect of the proteasome inhibitor MG-132 on NCC degradation. MDCK cells transiently expressing HA-NCC and full-length CHIP were incubated in either 10 μm MG-132 or dimethyl sulfoxide (DMSO) vehicle control. 4 h later, 100 μg/ml cycloheximide was added, and the samples were chased for 90 min. On the left are anti-HA immunoblots for NCC and anti-Myc immunoblots for CHIP; quantification of NCC degradation observed during the chase period is shown on the right. E, analysis of NCC polyubiquitination by CHIP. HEK293T cells transiently expressing HA-NCC with either full-length Myc-tagged CHIP or the Myc-tagged ΔUBox CHIP mutant were assayed 24 h post-transfection. Following 4 h of pretreatment with 10 μm MG-132, HA-NCC was immunoprecipitated from the lysates with anti-HA-conjugated agarose resin, and the immunoprecipitates were subjected to SDS-PAGE and immunoblotting (IB) with the indicated antibodies. In the total ubiquitin immunoblots, the immunoreactive signal corresponding to polyubiquitinated NCC is indicated in brackets. In both the ubiquitin and NCC immunoblots, a lower intensity signal corresponding to an NCC proteolytic fragment is indicated with an asterisk. An anti-Myc immunoblot from whole cell lysates (WCL, 15 μg; 5% input) is presented below the immunoprecipitations, demonstrating equivalent expression of both the full-length and mutant CHIP constructs. The figure is representative of four independent experiments. F, reciprocal TPR domain-dependent coimmunoprecipitation of NCC and CHIP. Left panels, whole cell lysates of HEK293T cells transiently expressing HA-NCC with Myc-CHIP, Myc-CHIP ΔTPR, or Myc-CHIP ΔUbox were immunoprecipitated with anti-HA-conjugated agarose resin and immunoblotted (IB) with anti-HA antibody (top panel) or anti-Myc antibody (bottom panel). Middle panels, the same whole cell lysates were immunoprecipitated with polyclonal anti-Myc antibody and probed with anti-HA antibody (top panel) or monoclonal anti-Myc antibodies (bottom panel). Right panels, 5% of the whole cell lysate inputs (15 μg/sample) for the immunoprecipitations were immunoblotted for HA-NCC (top panel) and Myc-CHIP (bottom panel) with the indicated antibodies.

Article Snippet: Polyclonal rabbit anti-mouse NCC antibody was purchased from Stressmarq.

Techniques: Expressing, Western Blot, Mutagenesis, Plasmid Preparation, Incubation, Transfection, Immunoprecipitation, SDS Page, Construct