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: Cerebellum (London, England)

Article Title: Developmental Upregulation of an Alternative Form of pcp2 with Reduced GDI Activity

doi: 10.1007/s12311-013-0529-0

Figure Lengend Snippet: Structure and sequence of the novel pcp2 transcript and protein. a Structure of the transcripts for long and short pcp2 (long and short refer to the protein and not to the mRNA). Gray and black boxes indicate previously known exons and the novel exon 3B, respectively. Lengths of exons and introns are indicated in base pairs. Arrows indicate primers used for RT-PCR. 1 L7sense, 2 L7anti, 3 L73Aanti. b Nucleotide sequence of exon 3B ( bold italic ) with intronic junctions. TER indicates the in-frame translation stop codon, and the gray - shaded box is the complement of the in situ hybridization probe. c Sequence of short and long pcp2 proteins. The gray - shaded box represents the GoLOCO motif. Sequences unique to either protein are framed , and predicted C-terminal phosphorylation sites are underlined with the phosphorylated serine being indicated by an upward arrow . d Computer-generated models of long and short pcp2. C-terminal domains that distinguish the two variants are indicated in orange . Red cylinders represent α -helical structures of the GoLOCO domain

Article Snippet: For the generation of recombinant glutathione S-transferase (GST)–pcp2 fusion proteins, cDNAs corresponding to short and longer transcript were cloned into the EcoRI and XhoI sites of the pGEX-4T-1 vector (GE Healthcare) in frame with the glutathione-S-transferase/thrombin cleavage site.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Generated

Journal: Cerebellum (London, England)

Article Title: Developmental Upregulation of an Alternative Form of pcp2 with Reduced GDI Activity

doi: 10.1007/s12311-013-0529-0

Figure Lengend Snippet: Developmental expression of the novel pcp2 transcript. Transcripts encoding short pcp2 protein were detected by transcript-specific RT-PCR at the indicated postnatal time points ( upper panel )

Article Snippet: For the generation of recombinant glutathione S-transferase (GST)–pcp2 fusion proteins, cDNAs corresponding to short and longer transcript were cloned into the EcoRI and XhoI sites of the pGEX-4T-1 vector (GE Healthcare) in frame with the glutathione-S-transferase/thrombin cleavage site.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cerebellum (London, England)

Article Title: Developmental Upregulation of an Alternative Form of pcp2 with Reduced GDI Activity

doi: 10.1007/s12311-013-0529-0

Figure Lengend Snippet: Localization of transcripts coding for the novel short pcp2 protein by in situ hybridization in adult C57BL/6 mice. a Dark field imaging of the cerebellar. g Granular cell layer, p Purkinje cell layer, m molecular layer. Bar 100 μm. b Control section. Bar 100 μm. c Bright field picture of Purkinje cells showing silver grains over cell bodies of Purkinje cells ( arrow heads ). Bar 100 μm. d Phase contrast image of mouse retinal layers. PE pigment epithelium, R&C rod and cone photoreceptors, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. Bar 100 μm. e Dark field image of a hybridized retina section. Bar 100 μm. f Control section. Bar 100 μm

Article Snippet: For the generation of recombinant glutathione S-transferase (GST)–pcp2 fusion proteins, cDNAs corresponding to short and longer transcript were cloned into the EcoRI and XhoI sites of the pGEX-4T-1 vector (GE Healthcare) in frame with the glutathione-S-transferase/thrombin cleavage site.

Techniques: In Situ Hybridization, Imaging

Journal: Cerebellum (London, England)

Article Title: Developmental Upregulation of an Alternative Form of pcp2 with Reduced GDI Activity

doi: 10.1007/s12311-013-0529-0

Figure Lengend Snippet: Influence of dark breeding on expression of pcp2. a Adult C57Bl/6 mice were kept in darkness for 60 h or 6 days after which tissues were processed for RT-PCR common for both transcripts. b P1 pups were kept in darkness with their mothers until tissue dissection at P15 and P19. Controls were kept under standard light conditions. The length of the amplimers is given in base pairs

Article Snippet: For the generation of recombinant glutathione S-transferase (GST)–pcp2 fusion proteins, cDNAs corresponding to short and longer transcript were cloned into the EcoRI and XhoI sites of the pGEX-4T-1 vector (GE Healthcare) in frame with the glutathione-S-transferase/thrombin cleavage site.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Dissection

Journal: Cerebellum (London, England)

Article Title: Developmental Upregulation of an Alternative Form of pcp2 with Reduced GDI Activity

doi: 10.1007/s12311-013-0529-0

Figure Lengend Snippet: Interaction with G protein α-subunits. a Interaction of Gα Subunits with short and long pcp2 protein. The indicated in vitro translated and S 35 -methionine labeled Gα subunits were incubated with recombinant GST fusions of AGS3, and short (SL7) and long pcp2 (L7), and GST alone, and then precipitates were analyzed by SDS-PAGE (see “ ” for details). Top panel the left lane is 5 % (Gαi2, Gαi3) or 10 % (Gαo, Gαi1) of input into the binding. Precipitated Gα subunits with each of the GST fusion proteins are shown in the subsequent lanes. The bottom panel shows Coomassie blue stained gel of the fusion proteins used in the binding experiments. Exposures are 24–48 h. b The binding of each Gα subunit to L7 and SL7 is shown relative to the AGS3 binding. c GTP binding to baculovirus-purified Gαo ( top ) and Gαi1 ( bottom ) in absence or presence of recombinant GST fusions of AGS3, short pcp2 (SL7), and long pcp2 (L7) (see “ ” for details). Specific binding of GTPγS to Gα subunits was determined in triplicate and set to 100 %. Results are the mean values ± SEM (Gαo, n = 8; Gαi1, n = 6 independent experiments). In comparison to Gαo alone, all three fusion proteins had small but significant reductions in GTPγS binding (SL7, p = 0.017; L7 and AGS3, p < 0.001). For Gαi1, p = 0.036 for SL7 and p < 0.001 for L7 and AGS3. The differences between short and long pcp2, and long pcp2 and AGS3 did not reach statistical significance, but short pcp2 was significantly different from AGS3 ( p = 0.003)

Article Snippet: For the generation of recombinant glutathione S-transferase (GST)–pcp2 fusion proteins, cDNAs corresponding to short and longer transcript were cloned into the EcoRI and XhoI sites of the pGEX-4T-1 vector (GE Healthcare) in frame with the glutathione-S-transferase/thrombin cleavage site.

Techniques: In Vitro, Labeling, Incubation, Recombinant, SDS Page, Binding Assay, Staining, Purification

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Human LDLR is proteolytically cleaved in its extracellular ligand binding domain by BMP1. ( A ) Schematic of the domain organisation of LDLR with a C-terminal FLAG tag showing the epitopes detected by the antibodies used in the study, antibody AF2148 (R&D Systems) raised against the entire ectodomain of LDLR, antibody Ab14056 (Abcam) raised against a recombinant protein fragment corresponding to amino acids 29–205 of LDLR) and antibody α-FLAG (Sigma-Aldrich) the anti-FLAG M2 antibody. TM, transmembrane domain; EGF, epidermal growth factor-like domain; F, FLAG epitope. ( B ) Immunoblot analysis with the indicated antibody of lysates and conditioned media samples from HepG2 cells expressing full-length FLAG-tagged human LDLR. Bands of interest were cropped from western blots of either media or lysate samples using each of the three antibodies. Images from separate western blots were combined but are separated by the dashed black line. Full blot images are presented in the Supplementary western blot dataset. ( C ) Schematic showing the proposed cleavage of the 160 kDa full-length (FL) LDLR to generate the 36–40 kDa NTF and 120 kDa CTF. ( D ) Immunoblot analysis of LDLR (antibody AF2148) without and with deglycosylation in liver biopsy samples from three separate individuals. The blot image was cropped to highlight the FL and CTF bands, full blot images are presented in the Supplementary western blot dataset. ( E ) Immunoblot analysis following incubation of rhLDLR (500 ng) with increasing amounts of rBMP1 at 37 °C for 1 h. ( F ) Immunoblot analysis following incubation of rhLDLR (500 ng) with rBMP1 (12.5 ng) in the absence or presence of the BMP1 inhibitor UK383367 (10 μM) at 37 °C for 1 h. ( G ) Immunoblot analysis following pre-incubation of rhLDLR (500 ng) in the absence or presence of LDL (5 µg), RAP (7.14pmol) or UK383367 (10 μM) for 30 min on ice followed by the addition of 12.5 ng rBMP1 and further incubation at 37 °C for 1 h. ( H ) Densitometric analysis of the Ab14056 immunoblot from ( C ) to determine the amount of FL and NTF as a percentage of total LDLR, data shown as mean ± SEM, statistical analysis using ANOVA with Tukey post-hoc pairwise analysis *p < 0.05, n = 3. For panels E–G, blot images were cropped to highlight the FL and CTF bands using the AF2148 antibody and the FL and NTF bands using the Ab14056 antibody due to different exposure times for visualisation of the FL and NTF bands. Full blot images are presented in the Supplementary western blot dataset.

Article Snippet: Membranes were incubated overnight at 4 °C in primary antibody diluted in 2% BSA in PBS-T: goat anti-human LDLR (AF2148), goat anti-human BMP1 (AF1927), goat anti-mouse LDLR (AF2255) (R&D systems), chicken anti-LDLR (Ab14056) (Abcam, Cambridge, UK) at a dilution of 1:1000, anti-FLAG M2 antibody (Sigma-Aldrich) at a dilution of 1:2500 and anti-actin antibody (Sigma-Aldrich) at a dilution of 1:5000.

Techniques: Ligand Binding Assay, FLAG-tag, Recombinant, Western Blot, Expressing, Incubation

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Cleavage of human LDLR by BMP1 reduces the binding and cellular uptake of LDL. ( A ) Immunoblot analysis following incubation of rhLDLR-His (5 μg) with rBMP1 (200 ng) at 37 °C for 1 h. The blot image was cropped to highlight the FL and CTF bands, full blot images are presented in the Supplementary western blot dataset. ( B ) Dot blot of BODIPY ® -LDL (1 μg/ml) binding to rhLDLR-His digested with BMP1 (from A), BACE1-His and PCSK9-FLAG (all 0.5 μg). Membranes were re-probed with anti-His and anti-FLAG antibodies. ( C ) Densitometric analysis of the BODIPY-LDL dot blot from ( B ) showing the percentage binding to rhLDLR-His in the absence or presence of rBMP1. Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, n = 4. ( D ) Immunoblot analysis of LDLR with actin as a loading control in lysates from CHO-A7 cells expressing either full-length (FL) LDLR or 120 kDa CTF, or the empty vector (EV). The blot images were cropped to highlight the FL and CTF bands using the AF2148 and anti-FLAG antibodies and actin using the AC15 antibody. The full blot images are presented in the Supplementary western blot dataset. ( E ) Immunoblot analysis of LDLR from lysates (L) and from the cell-surface fraction (S) in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF followed by incubation with either vehicle (DMSO) or EZ-Link™ Sulfo-NHS-SS-Biotin (0.5 mg/ml) for 20 min on ice and immunoprecipitation with Streptavidin-agarose beads for 3 h at 4 °C. The blot image was cropped to highlight the FL and CTF bands using the AF2148 antibody. The full blot image is presented in the Supplementary western blot dataset. ( F ) Immunofluorescence microscopy images showing LDLR expression (green) and Dil-LDL binding (red) in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF followed by incubation with Dil-LDL for 30 s on ice. ( G ) Quantification of Dil-LDL mean intensity in either the FL LDLR or 120 kDa CTF expressing cells from ( F ), data shown as mean ± SEM, statistical analysis performed using a Student’s T-test *p < 0.05, n = 7. ( H ) BODIPY ® -LDL uptake in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF. Data expressed as percentage of FL following subtraction of empty vector uptake as a baseline and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, *p < 0.05, n = 4.

Article Snippet: Membranes were incubated overnight at 4 °C in primary antibody diluted in 2% BSA in PBS-T: goat anti-human LDLR (AF2148), goat anti-human BMP1 (AF1927), goat anti-mouse LDLR (AF2255) (R&D systems), chicken anti-LDLR (Ab14056) (Abcam, Cambridge, UK) at a dilution of 1:1000, anti-FLAG M2 antibody (Sigma-Aldrich) at a dilution of 1:2500 and anti-actin antibody (Sigma-Aldrich) at a dilution of 1:5000.

Techniques: Binding Assay, Western Blot, Incubation, Dot Blot, Control, Expressing, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Genetic knockdown of BMP1 decreases the proteolytic cleavage of LDLR. ( A ) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated either with siRNA against BMP1 (Dharmacon) or with a non-targeting control siRNA. Blot images were cropped to highlight the FL and CTF bands in cell lysates using the AF2148 antibody and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( B ) Densitometric analysis of the NTF from ( A ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, n = 6. ( C ) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated either with siRNA against BMP1 (Ambion) or with a non-targeting control siRNA. Blot images were cropped to highlight the FL and CTF bands in cell lysates using the AF2148 antibody and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( D ) Relative expression of BMP1 mRNA in HepG2 cells treated with siRNA against BMP1 (Ambion) or with a non-targeting control. Data expressed relative to control and shown as mean ± SEM, statistical analysis performed using an independent t-test with Welch’s correction for two sample comparison **p < 0.01, n = 3. ( E ) Immunoblot analysis of LDLR and BMP1 in cell lysates from HepG2 cells treated with siRNA against BMP1 (Ambion) or with a non-targeting control siRNA and then transfected either with an empty vector, BMP1-FLAG or BMP1 E 214 Q-FLAG constructs. Blot images were cropped to highlight the FL and CTF band of LDLR using the AF2148 antibody and BMP1 using the AF1927 antibody. Full blot images are presented in the Supplementary western blot dataset. ( F ) Densitometric analysis of FL LDLR and the CTF from ( E ). Data expressed as percentage of NT siRNA control (not shown in graph) and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis **p < 0.01, ***p < 0.001, n = 3.

Article Snippet: Membranes were incubated overnight at 4 °C in primary antibody diluted in 2% BSA in PBS-T: goat anti-human LDLR (AF2148), goat anti-human BMP1 (AF1927), goat anti-mouse LDLR (AF2255) (R&D systems), chicken anti-LDLR (Ab14056) (Abcam, Cambridge, UK) at a dilution of 1:1000, anti-FLAG M2 antibody (Sigma-Aldrich) at a dilution of 1:2500 and anti-actin antibody (Sigma-Aldrich) at a dilution of 1:5000.

Techniques: Knockdown, Western Blot, Control, Expressing, Comparison, Transfection, Plasmid Preparation, Construct

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Pharmacological inhibition of BMP1 decreases the proteolytic cleavage of LDLR and increases the cellular uptake of LDL. ( A ) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated with inhibitor UK383367 (10 μM) for 18 h. Blot images were cropped to highlight the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( B ) Densitometric analysis of the NTF from ( A ). Data shown as mean ± SEM, ***p < 0.001, n = 3. ( C ) BODIPY ® -LDL uptake in HepG2 cells treated with or without inhibitor UK383367 (10 μM) for 6 h. Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test **p < 0.01, n = 3. ( D ) Representative data from flow cytometry showing the change in cell surface fluorescence of LDLR between HepG2 cells treated with inhibitor UK383367 (10 μM) for 18 h (blue line) and untreated cells (red line); primary antibody (Ab14056) only (grey line). ( E ) Quantitation of the cell surface expression of LDLR in HepG2 cells treated with or without UK383367 as analysed by flow cytometry in ( D ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test **p < 0.01, n = 3.

Article Snippet: Membranes were incubated overnight at 4 °C in primary antibody diluted in 2% BSA in PBS-T: goat anti-human LDLR (AF2148), goat anti-human BMP1 (AF1927), goat anti-mouse LDLR (AF2255) (R&D systems), chicken anti-LDLR (Ab14056) (Abcam, Cambridge, UK) at a dilution of 1:1000, anti-FLAG M2 antibody (Sigma-Aldrich) at a dilution of 1:2500 and anti-actin antibody (Sigma-Aldrich) at a dilution of 1:5000.

Techniques: Inhibition, Western Blot, Control, Flow Cytometry, Fluorescence, Quantitation Assay, Expressing

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Cleavage of LDLR requires the catalytic activity of BMP1 and human but not murine LDLR is proteolytically cleaved by BMP1 in vivo . ( A ) Immunoblot analysis of BMP1 in conditioned media and of LDLR in cell lysates and conditioned media from SW13 cells expressing wild-type (WT) or mutated (E 214 Q) BMP1. Blot images were cropped to highlight BMP-1 bands in conditioned media and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( B ) Densitometric analysis of FL and the NTF from ( A ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis *p < 0.05, **p < 0.01, n = 4. ( C ) BODIPY ® -LDL uptake in SW13 cells expressing either WT or E 214 Q BMP1. Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis **p < 0.01, n = 3. ( D ) Immunoblot analysis of rhLDLR (500 ng) (top panel) and recombinant mouse LDLR (500 ng) (bottom panel) following incubation with rBMP1 (100 ng) for 1 h. Blot images were cropped to highlight the FL and CTF bands in human LDLR using the AF2148 antibody and the FL band only in mouse LDLR using AF2255 antibody. Full blots are not available for this figure. ( E ) Immunoblot analysis of LDLR in lysates from HepG2 cells expressing either wild-type (WT) or mutated (D 172 V) LDLR and incubated with or without inhibitor UK383367 (10 μM) for 18 h. The blot image was cropped to highlight the FL and CTF bands using the anti-FLAG antibody. Full blot images are presented in the Supplementary western blot dataset. ( F ) Densitometric analysis of FL -LDLR and the 120 kDa CTF from ( E ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis **p < 0.01, n = 3. ( G ) Immunoblot analysis of human LDLR (top panel) and mouse LDLR (bottom panel) in liver lysates from either wild-type LDLR +/+ or transgenic LDLR h/+ mice. Blot images were cropped to highlight the FL and CTF bands using the AF2148 antibody and the FL band only in mouse LDLR using AF2255 antibody. Full blot images are presented in the Supplementary western blot dataset.

Article Snippet: Membranes were incubated overnight at 4 °C in primary antibody diluted in 2% BSA in PBS-T: goat anti-human LDLR (AF2148), goat anti-human BMP1 (AF1927), goat anti-mouse LDLR (AF2255) (R&D systems), chicken anti-LDLR (Ab14056) (Abcam, Cambridge, UK) at a dilution of 1:1000, anti-FLAG M2 antibody (Sigma-Aldrich) at a dilution of 1:2500 and anti-actin antibody (Sigma-Aldrich) at a dilution of 1:5000.

Techniques: Activity Assay, In Vivo, Western Blot, Expressing, Control, Recombinant, Incubation, Transgenic Assay

Journal: PLoS Genetics

Article Title: Ataxin-2 Regulates RGS8 Translation in a New BAC-SCA2 Transgenic Mouse Model

doi: 10.1371/journal.pgen.1005182

Figure Lengend Snippet: (A) SH-SY5Y whole cell extracts expressing Flag-ATXN2-Q22 or Flag-ATXN2-Q108 were subjected to immunoprecipitation with a mAb to the Flag tag. After washing the beads with buffer (200 mM NaCl), bound protein-RNA complexes were eluted by Flag peptide competition. IP products were divided equally into two parts and subjected to Western blot and RT-PCR analyses to identify ATXN2 interacting proteins and RNAs. Western blot analyses of the eluted proteins show co-IP of PABPC1 and DDX6, known ATXN2 interactors. RT-PCR analyses of the second aliquot show that both Flag-ATAXN2-Q22 and Flag-ATAXN2-Q108 immunoprecipitate RGS8 mRNA but not GAPDH mRNA. ATXN2-Q108 shows differential binding toward RGS8 mRNA when compared with ATXN2-Q22. (B) Interaction of ATXN2 with RGS8 mRNA determined by qRT-PCR. Synthesized cDNAs from the second aliquot of IP products (A) were subjected to qRT-PCR analyses. Interaction of RGS8 mRNA with ATXN2-Q108 was significantly reduced when compared with ATXN2-Q22. Data are mean ± SD, n = 3 independent experiments. **p<0.01. (C-D) Mutant ATXN2 represses RGS8 synthesis in vitro . First, cDNA plasmids of LacZ (control) and Flag-tagged ATXN2-Q22 or -Q108 were added to rabbit reticulocyte lysate mixture and proteins synthesized for 2 hrs. RGS8 cDNA plasmid was added to each translational reaction with fresh rabbit reticulocyte lysate and incubated further for 4 hrs. The synthesized RGS8 protein from each translational product was analyzed by SDS-PAGE followed by Western blot analyses. ATXN2-Q108 reduces RGS8 synthesis significantly when compared with ATXN2-Q22 (C). Quantification of RGS8 on Western blots, data are mean ± SD, n = 3 independent experiments. **p<0.01, Student’s t-test) (D). The blot represents one of three independent immunoprecipitation experiments.

Article Snippet: ATXN2 mAb [(1:3000), BD Biosciences Inc.; cat# 611378], 5TF1-1C2 mAb [(1:3000), Millipore Inc.; #MAB1574], RGS8 rabbit polyclonal Ab [(1:3000), Novus Biologicals; #NBP2-20153], Calbindin-D-28K mAb [(1: 5000), Sigma Inc.; cat# C9848], PCP2 mAb [(1: 5000), Santa Cruz Inc.; cat# sc-137064], DDX6 rabbit polyclonal Abs [(1:4000), Santa Cruz Inc.; cat# sc-27127-R], PABPC1 mAb [(1:4000), Santa Cruz Inc.; cat# sc-27127-R], CUG-BP1 mAb [(1:4000), Santa Cruz Inc.; cat# sc-20003], Flag M2 mAb [(1:10,000), Sigma Inc.; cat# F3165], GFP mAb [(1:3000), Santa Cruz Inc.; cat# sc-9996] and MYC mAb conjugated with HRP [(1:5000), Invitrogen Inc.; cat# A3858].

Techniques: Expressing, Immunoprecipitation, FLAG-tag, Western Blot, Reverse Transcription Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Binding Assay, Quantitative RT-PCR, Synthesized, Mutagenesis, In Vitro, Control, Plasmid Preparation, Incubation, SDS Page

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: (A) Ataxin-1 and BACE1 levels in 6-month-old Atxn1154Q/+ and WT mice. Arrow and dashed arrow indicate the positions of WT (2Q) and mutant (154Q) Ataxin-1, respectively. In bar, number of mice analyzed. *p < 0.05, **p < 0.01, ***p < 0.001, t-test. (B) BACE1 immunolabeled cerebellum of 1.5-, 4.5-, and 8-month-old Atxn1154Q/+ mice. *, molecular layer. Bar = 200 μm. (C) Bace1 mRNA levels in the cortex and cerebellum. Relative value of 100 is given to the cortex and cerebellum of WT at each age group. (D) Hippocampus of Atxn1154Q/+ and WT mice immunolabeled for BACE1. Arrows, BACE1-immunopositive mossy fiber ends. Bar = 200 μm. (E) Quantification of BACE1-immunoreactive area near CA2. In bar, number of brain section analyzed. 1–2 sections/mouse. (F) Hippocampal CA1/CA2/CA3 regions of 8-month-old mice immunolabeled for Ataxin-1 and BACE1. Arrow head, punctate staining of CA1 neurons by Ataxin-1 and DAPI. *, outward shape of CA2 region. Arrows in inset, BACE1 immunoreactivity (yellow signal) in the neuronal soma. Bar = 100 μm. (G) Immunofluorescence signals of Ataxin-1 and synaptoporin, a presynaptic marker. Arrows, synaptoporin+ presynaptic terminals. Insets, high resolution images without Ataxin-1 signal. (H) Hippocampal regions immunolabeled for PCP4 and NeuN, a neuronal marker. Bracket, CA2; *, stratum lucidum; arrow, granular neurons. (I) Counts of PCP4+ neurons and NeuN+ neurons in CA2 regions of 6-9 months-old mice. In bar, numbers of brain section analyzed. 2–4 sections/mouse. (J) Quantification of PCP4 immunoreactivity of CA2 neurons. 1–2 sections/mouse. (K) RGS14 immunoreactivity in 6 month-old mice. Arrows, RGS14+ dendrites of CA2 neurons; *, RGS14+ axons. Bar = 100 μm. (L) Dentate gyrus of 4.5-month-old mice immunolabeled for DCX. Arrows, DCX+ neurons. *, background DCX immunoreactivity. Right: counts of total and projecting DCX+ cells per dentate gyrus. n = 4. See also Figures S6 and S7.

Article Snippet: Primary antibodies used for immunostaining are Ataxin-1 (76/8, NeuroMab), Aβ (3D6, Eli Lilly), BACE1 (D10E5, Cell Signaling), DCX (SC-8066, Santa Cruz), GFAP (Z0334, Dako), Iba1 (019-19741, Wako), Ki-67 (DRM004, Acris Antibodies), NeuN (RPCA-FOX3, EnCor Biotechnology), PCP4 (HPA005792, Atlas Antibodies), RGS14 (75-170, Neuromab), STEP (4396, Cell Signaling), synaptoporin (102 002, Synaptic Systems).

Techniques: Mutagenesis, Immunolabeling, Staining, Immunofluorescence, Marker

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used for immunostaining are Ataxin-1 (76/8, NeuroMab), Aβ (3D6, Eli Lilly), BACE1 (D10E5, Cell Signaling), DCX (SC-8066, Santa Cruz), GFAP (Z0334, Dako), Iba1 (019-19741, Wako), Ki-67 (DRM004, Acris Antibodies), NeuN (RPCA-FOX3, EnCor Biotechnology), PCP4 (HPA005792, Atlas Antibodies), RGS14 (75-170, Neuromab), STEP (4396, Cell Signaling), synaptoporin (102 002, Synaptic Systems).

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Staining, Expressing, Knock-In, SYBR Green Assay, Clone Assay, Mutagenesis, Software, Microscopy, Real-time Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: A novel antiviral lncRNA EDAL shields a T309 O -GlcNAcylation site to promote EZH2 degradation

doi: 10.1101/824813

Figure Lengend Snippet: A. N2a cells were transfected with pcDNA3.1 or pcDNA-EDAL for 12 h and then infected with RABV at MOI 1 for 48 h. Total RNA was isolated and subjected to RNA-seq analysis ( n =2; 2 fold change (FC) and 0.01 p-value ). B. N2a cells were transfected with pcDNA3.1 or pcDNA-EDAL for 48 h and then ChIP-seq analysis was performed. Volcano plot showed the peaks enriched in negative control (NC) cells and EDAL overexpression cells. X axis was the log2 ratio of EDAL versus NC signals for each peak, and Y axis was the significance of the differences (−log10 ( P-values )). C. Six up-regulated and loss of H3K27me3 mark genes were cloned into the mammalian expression vector pCAGGS and overexpressed in N2a cells. At 12 h post transfection, the cells were infected with RABV for 48 h at MOI 0.01, and virus titers in the supernatant were measured. D. N2a cells were transfected with pCAGGS- Pcp4l1 (pC- Pcp4l1 ) at indicated dose for 12 h, and then infected with RABV at MOI 0.01. At 48 hpi, the virus load in the cell supernatant was measured. PCP4L1 expression level was analyzed by Western blotting. E. pcDNA-RABV-N, pcDNA-RABV-P together with pCAGGS or pC-Pcp4l1-flag was transfected into N2a cells for 48 h. The level of RABV-N protein and RABV-P protein was analyzed by Western blotting and normalized to GAPDH. F. N2a cells were transfected with pCAGGS- Pcp4l1 (pC- Pcp4l1 ) for 12 h, and then infected with VSV at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. G. N2a cells were transfected with pC- Pcp4l1 for 24 h, and then infected with SFV at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. H. N2a cells were transfected with pC- Pcp4l1 for 24 h, and then infected with HSV-1 at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. I. Sequencing profile of Pcp4l1 for ChIP-seq. The two tracks show H3K27me3 signals for pcDNA3.1 and pcDNA-EDAL samples after removing input background. The brown rectangle indicates the predicted promoter region of Pcp4l1 . J. N2a cells were transfected with pcDNA-EDAL or pcDNA3.1 for 48 h, and then ChIP-qPCR were performed with H3K27me3 antibody in the promoter region of Pcp4l1 . K. N2a cells were treated with 4 µM gsk126 or DMSO (mock) for 48 h and Pcp4l1 mRNA level was analyzed by qPCR. L. Proposed model for EDAL-induced EZH2 lysosomal degradation, and the potential subsequent impact on EZH2-mediated epigenetic silencing of Pcp4l1 . Statistical analysis of grouped comparisons was carried out by student’s t test(**P<0.01; ***P<0.001). Bar graph represents means ± SD, n = 3.

Article Snippet: The primary antibodies were against RABV N protein (prepared by our lab, 1:5000), H3K27me3 (Abclonal Technology, Wuhan, China, A2363, 1:2000), H3 (Abclonal Technology, A2348, 1:2000), EZH2 (CST, #5246, 1:2000), Flag tag (MBL, M185-3L, 1:10000), HA tag (MBL, M180-3, 1:10000), PCP4L1 (ProteinTech, 25933-1-AP, 1:2000) or GAPDH (ProteinTech, 60004-1-Ig, 1:5000).

Techniques: Transfection, Infection, Isolation, RNA Sequencing Assay, ChIP-sequencing, Negative Control, Over Expression, Clone Assay, Expressing, Plasmid Preparation, Western Blot, Sequencing

Journal: Molecular Cell

Article Title: Mapping the invisible chromatin transactions of prophase chromosome remodeling

doi: 10.1016/j.molcel.2021.12.039

Figure Lengend Snippet:

Article Snippet: Plasmid: 3Xsuperfolding GFP , Addgene , 75385.

Techniques: Recombinant, Multiplex sample analysis, dsDNA Assay, Transfection, Mass Spectrometry, Western Blot, Microscopy, Expressing, Sequencing, Plasmid Preparation, Knock-In, Construct, Software