Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and β-actin was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Sequencing, Standard Deviation, Synthesized, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) Plasmid encoding Flag-tagged catalytic-deficient nsp15 H223A, H238A, oligomerization-deficient nsp15 D285A, D315A, wild type nsp15, and the vector PXJ40, were each co-transfected with plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-IRF7, or EGFP into DF-1 cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands was analysed by Image J, normalized to the signal of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 transfected cells. (B) DF-1 (C) Vero and H1299 cells, were transfected with the plasmid encoding wild type or mutated nsp15 and treated with puromycin (5 µg/ml) for 1 h at 23 h post-transfection (h.p.t), to label the de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin signal along the white line (from a to b) is indicated in the right panel (histogram plot).

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Plasmid Preparation, Transfection, Western Blot, Synthesized, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: The plasmid encoding wild type or catalytic-deficient nsp15 from the indicated coronaviruses was co-transfected with the plasmid encoding EGFP or IBV N into Vero cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands of EGFP or IBV N were analysed by Image J, normalized to the signal of β-actin and presented relative to the PXJ40 group.

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Plasmid Preparation, Transfection, Western Blot

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: Plasmid encoding wild type or catalytic-deficient IBV nsp15 and reporter plasmid encoding IBV N or IBV M, or luciferase DNA, were co-incubated with Rabbit Reticulocyte Lysate for 1 h followed by Western blot analysis or luciferase assay. Density of the bands corresponding to the reporter proteins was normalized to the signal of β-actin and presented relative to the sample transfected with the empty vector PXJ40.

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Plasmid Preparation, Luciferase, Incubation, Western Blot, Transfection

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) DF-1 cells or (B) H1299 cells were infected with IBV-WT or rIBV-nsp15H1238A at an MOI of 1. At 6, 12, 24 h.p.i., cells were treated with puromycin (5 µg/ml) for 1 h, followed by western blot analysis to detect puromycin-labelled de novo peptides, IBV-N protein, and β-actin. Density of the puromycin labelled proteins was normalized to the signal of β-actin. Ratio of the puromycin–labelled de novo peptides of the infected cells (+) to that of the uninfected cells (-) at the same time h.p.i. is shown. (B) H1299 cells were infected as described above followed by dot blot analysis to detects dsRNA and western blot analysis to detect p-PKR, PKR, p-eIF2α, eIF2α.

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Infection, Western Blot, Dot Blot

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) DF-1 cells were transfected with a plasmid coding Flag-tagged nsp15 (PXJ40F-nsp15). At 24 h.p.t, indirect immunofluorescence was performed with a chicken anti-Flag-tag antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV-WT or rIBV-nsp15-H238A at an MOI=1. At 18 h.p.i, indirect immunofluorescence was performed with a mouse anti-IBV-nsp15 monoclonal antibody (red), a rabbit anti-IBV-nsp12 polyclonal antibody (green) and the nuclei were stained with DAPI (blue). (C) Vero cells were infected with IBV or rIBV-nsp15-H238A with an MOI of 1. At 18 h.p.i., cells were treated with 100 μg/mL cycloheximide (CHX) for 15 min at 37°C and subjected to 7–47% sucrose density gradient ultracentrifugation (38,000 rpm for 3 h), and the fractions were analysed by Western blot to detect nsp15, Rsp6, eIF4E, and β-actin (left panel).

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, FLAG-tag, Staining, Infection, Western Blot

Journal: bioRxiv

Article Title: Myosin VIII and XI isoforms interact with Agrobacterium VirE2 protein and help direct transport from the plasma membrane to the perinuclear region during plant transformation

doi: 10.1101/2023.03.06.531343

Figure Lengend Snippet: (A) Agrobacterium -mediated transient transformation assays were conducted on roots of four independent transgenic lines of wild-type (Lines 2, 4, 5 and 8) or (B) the myosin VIII-1/2/a/b mutant (Lines 2, 3, 12, and 13). Root segments were inoculated with 10 6 , 10 7 , or 10 8 cfu/ml of the virE2 mutant strain A. tumefaciens At1879 containing the T-DNA binary vector pBISN2. Plants were treated with β-estradiol (gray bars) or control solution (black bars) for VirE2-Venus expression 24 hr before infection. The percentage of roots showing GUS activity was calculated as in . A total of 10-15 plants from each line and >100 segments per plant were tested for transient transformation. Values given are means ± SE. Asterisks indicate significant differences compared to uninduced plants. [ t-test , * P < 0.05; ** P < 0.01]. (C) Confocal images showing aggregation of VirE2-Venus proteins in transgenic roots of either wild-type (top panel) or myosin VIII 1/2/a/b mutant backgrounds (bottom panel). (D) Quantitative analysis of the size of VirE2-Venus aggregates and the percentage of the cellular area occupied by the aggregates. Image J was used for analysis. The average VirE2-Venus aggregate size was 2.0±0.3 μm 2 in wild-type roots, and 6.2±0.6 μm 2 in myosin VIII-1/2/a/b quadruple mutant roots. (E) Western blot detection of VirE2-Venus proteins expressed in transgenic plants of either the Col-0 or the myosin VIII-1/2/a/b quadruple myosin mutant background. Mouse anti-GFP antibody was used to detect VirE2-Venus protein expressed after induction of individual transgenic lines. The house-keeping protein phosphoenolpyruvate carboxylase (PEPC) was detected using a rabbit anti-PEPC antibody and served as an internal control.

Article Snippet: Protein samples were loaded onto 10% SDS polyacrylamide gels, subjected to electrophoresis (60 Volts 1.5 hrs for the stacking gel and 100 Volts 4.5 hrs for the separating gel), then either stained with Coomassie blue or subjected to western blot analysis using a 1:1000 dilution of mouse anti-GFP or mouse anti-Myc antibodies (Cell Signaling Technology, USA).

Techniques: Transformation Assay, Transgenic Assay, Mutagenesis, Plasmid Preparation, Expressing, Infection, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Myosin VIII and XI isoforms interact with Agrobacterium VirE2 protein and help direct transport from the plasma membrane to the perinuclear region during plant transformation

doi: 10.1101/2023.03.06.531343

Figure Lengend Snippet: VirE2-Venus or Venus proteins were incubated with the indicated myc-tagged myosin CBD, VIP1, or Lamin C. Following binding to anti-GFP beads, proteins were eluted and subjected to Western blot analysis using anti-myc antibodies. Coomassie stained gels are shown in panels A , C , and E , and immunoblots in panels B , D , and F . VirE2-Venus interacts with the CBDs of myosins VIII-2, VIII-A, VIII-B, and XI-K, and with VIP1 (panels B , D , and F ), but not with the CBD of myosin VIII-1 or with Lamin C (panels D and F ). Venus, as a negative control, does not interact with any myc-tagged protein.

Article Snippet: Protein samples were loaded onto 10% SDS polyacrylamide gels, subjected to electrophoresis (60 Volts 1.5 hrs for the stacking gel and 100 Volts 4.5 hrs for the separating gel), then either stained with Coomassie blue or subjected to western blot analysis using a 1:1000 dilution of mouse anti-GFP or mouse anti-Myc antibodies (Cell Signaling Technology, USA).

Techniques: Incubation, Binding Assay, Western Blot, Staining, Negative Control

Journal: bioRxiv

Article Title: Myosin VIII and XI isoforms interact with Agrobacterium VirE2 protein and help direct transport from the plasma membrane to the perinuclear region during plant transformation

doi: 10.1101/2023.03.06.531343

Figure Lengend Snippet: (A) Protein extracts from roots of transgenic Arabidopsis plants expressing VirE2-Venus, or Venus, and the indicated myc-tagged myosin CBD transgenes were incubated with anti-GFP beads, the bound proteins eluted, and subjected to Western blot analysis using anti-myc antibodies. Myc-tagged VIP1 was used as a positive control. (B) Root extracts expressing VirE2-Venus or Venus and myc-tagged myosin VIII-2 CBD were incubated with anti-GFP beads, the bound proteins eluted, and subjected to Western blot analysis using anti-myc antibodies.

Article Snippet: Protein samples were loaded onto 10% SDS polyacrylamide gels, subjected to electrophoresis (60 Volts 1.5 hrs for the stacking gel and 100 Volts 4.5 hrs for the separating gel), then either stained with Coomassie blue or subjected to western blot analysis using a 1:1000 dilution of mouse anti-GFP or mouse anti-Myc antibodies (Cell Signaling Technology, USA).

Techniques: Transgenic Assay, Expressing, Incubation, Western Blot, Positive Control

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: PERK recruits the lipid transfer protein E-Syt1 at the EMCS. (A) Representative immunoblot for myc, GFP, and CNX after GFP pull-down showing PERK-E-Syt1 interaction in HEK293-T cells transiently co-transfected with myc-tagged PERK full-length (FL) or myc-tagged PERK kinase dead mutant (PERKK618A) and with eGFP-empty vector or eGFP-tagged E-Syt1. Untransfected cells are shown as negative control. In the eGFP-E-Syt1 transfected cells, a residual GFP signal is still visible as upper band (blue arrow) above PERK (red arrow) in the anti-myc panel. (B) Quantification of the PERK-E-Syt1 interaction normalized on GFP-pulled down and relative to control condition (PERK-myc). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (C) Representative immunoblot for eGFP and PERK showing the interaction of PERK and E-Syt1 in HEK293-T cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 pulled down. (D) Representative electron micrograph of ultrathin cryosections of HeLa cells transfected with eGFP-E-Syt1 and immunogold stained with anti-GFP (15 nm gold particles) and anti-PDI (10 nm gold particles). Black arrow denotes E-Syt1 detection at the sites of juxtaposition between the ER and the mitochondria membranes, while PDI, a general ER marker, remains in the ER lumen. Scale bar, 500 nm. (E) Relative quantification of the cellular distribution of E-Syt1 in the ER, EMCS (MAMs), and plasma membrane (PM). The values plotted are the mean ± SEM ( n = 16 cellular profiles). (F) Representative immunoblot for IP3R3, PERK, E-Syt1, CNX, VDAC1 and CYTC from total lysates, crude mitochondrial fraction (mito crude), purified mitochondrial fraction (mito pure) and MAM fraction of shCTR and shPERK HeLa cells. (G and H) Quantification of E-Syt1 level at MAMs (G) and total lysate (H) normalized on CNX levels and relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (I) Representative immunoblot for PERK in shCTR, shPERK, and shPERK + PERKK618A HeLa cells. ACTIN serves as loading control. (J) Representative images from eGFP-E-Syt1 transiently transfected and co-stained with MitoTracker Far Red in shCTR, shPERK and shPERK + PERKK618A HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (K) Colocalization analysis of E-Syt1 and MitoTracker Far Red in shCTR, shPERK and shPERK + PERKK618A HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 26, n = 26, and n = 25 for shCTR, shPERK, and shPERK + PERKK618A respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (L) Abundance of PC, PI, PE from purified mitochondrial fractions of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: .

Article Snippet: Mouse monoclonal anti ACTIN, Sigma-Aldrich, A5441; Mouse monoclonal anti c-myc, Sigma-Aldrich, M4439; Rabbit polyclonal anti Calnexin, Enzo, ADI-SPA-865-F; Mouse monoclonal anti CYTC, BD Bioscience, 556433; Rabbit DyLight 680, Thermo Fisher Scientific, 35569; Mouse DyLight 680 Thermo Fisher Scientific, 35519; Mouse DyLight 800 Thermo Fisher Scientific, 35521; Rabbit DyLight 800 Thermo Fisher Scientific, 35571; Mouse monoclonal anti eIF2α, Cell Signaling, 2103S; Rabbit polyclonal anti-E-Syt1, Sigma-Aldrich, HPA016858; Rabbit polyclonal anti-GFP, Cell Signaling, 2555S; Mouse monoclonal anti-GFP,Life technologies, A11122; HRP Mouse Bioké, Cell Signaling, 7076; HRP Rabbit Bioké, Cell Signaling, 7074; Mouse monoclonal anti IP3R3, BD Bioscience, 610312; Rabbit polyclonal anti-PERK, Cell signaling, 3192S; Rabbit polyclonal anti PERK, Cell signaling, 5683S; Rabbit monoclonal anti Phospho-eIF2α (Ser51), Cell signaling, 3597S; Rabbit polyclonal anti PDI Genetex, GTX30716; Mouse monoclonal anti PSD, Santa Cruz, sc-390070; Rabbit polyclonal anti, PSS1 (B-5), Santa Cruz, sc-515376; Rabbit polyclonal anti PSS2, Sigma-Aldrich, SAB1303408; Rabbit polyclonal VDAC1, Cell Signaling, 4866S; Rabbit polyclonal VDAC1, Abcam, ab15895; Veriblot antibody Abcam, ab131366.

Techniques: Western Blot, Transfection, Mutagenesis, Plasmid Preparation, Negative Control, Staining, Marker, Purification

Journal: PLoS ONE

Article Title: Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

doi: 10.1371/journal.pone.0068491

Figure Lengend Snippet: (A) The subcellular localization of GFP-hSHP in 293T cells was observed under a fluorescence microscope, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 100 µm, 50 µm (insets). (B) The N-terminal domain of SHP is primarily responsible for the interaction with HNF4α protein. Left, Diagram showing the deletion mutation constructs of GFP-mShp. Right, 293T cells were transfected with GFP-mSHP deletion constructs (4 µg, 6 cm plate) along with HA-HNF4α (4 µg, 6 cm plate) expression vector. Anti-HA agarose was used to immuneprecipitate HNF4α, and the protein levels of various GFP-mSHP truncation mutants and HNF4α were detected by Western blots using anti-GFP or anti-HA antibodies, respectively. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.

Article Snippet: The VDAC antibody (#4661), COX IV antibody (#4850), Hsp60 antibody (#4870), Cyto c antibody (#4280), PARP antibody (#9532), Bcl2 antibody (#2870), myc antibody (#2278), GFP antibody (#2955), GST antibody(#2625), and HNF4α antibody (#3113) were purchased from Cell Signaling.

Techniques: Fluorescence, Microscopy, Immunofluorescence, Mutagenesis, Construct, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

doi: 10.1371/journal.pone.0068491

Figure Lengend Snippet: The subcellular localization of GFP-mSHP full length and deletion mutants in 293T cells was observed by fluorescence microscopy, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 50 µm. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.

Article Snippet: The VDAC antibody (#4661), COX IV antibody (#4850), Hsp60 antibody (#4870), Cyto c antibody (#4280), PARP antibody (#9532), Bcl2 antibody (#2870), myc antibody (#2278), GFP antibody (#2955), GST antibody(#2625), and HNF4α antibody (#3113) were purchased from Cell Signaling.

Techniques: Fluorescence, Microscopy, Immunofluorescence