Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Schematic representation of STIM1 mutants with deletions in the 551-685 region. The specific sequences deleted between the amino acids 551 and 685 for each of the generated STIM1 mutants are shown. This diagram was created with BioRender.com. ( B ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ643-677)-GFP (mutant 7), STIM1(Δ668-674)-GFP (mutant 8), STIM1(Δ672-685)-GFP (mutant 9), or GFP (empty vector) as a control. Co-precipitation of GRP75 was assessed by immunoblotting. WCL (3 µg) from cells expressing STIM1-GFP was loaded as a positive control. Total immunoprecipitated GFP was used as a loading control. Total levels of STIM1 and GRP75 in WCL (30 μg protein/lane) were analyzed by immunoblotting (Fig. ). ( C ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ551-611)-GFP (mutant 5), STIM1(Δ582-642)-GFP (mutant 6), STIM1(Δ672-685)-GFP (mutant 9), or GFP only as a control. Other conditions as stated for ( B ). Total levels of STIM1 and GRP75 in WCL were analyzed by immunoblotting (30 μg protein/lane) (Fig. ). ( D ) STIM1-KO HEK293 cells inducibly expressing full-length (FL) STIM1-GFP or STIM1(Δ551-611)-GFP were fixed and incubated with rabbit anti-GRP75 and/or sheep anti-GFP antibodies. Negative controls consisted of cells incubated with single primary antibodies. Scale bar = 10 μm. ( E ) Quantification of the interactions per cell detected by PLA in ( D ). The number of cells evaluated is indicated in parentheses, and the mean is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001 and p = 0.5354 (n.s.). ( F ) STIM1-KO HEK293 cells expressing STIM1-GFP (full-length, FL) or STIM1(Δ551-611)-GFP were incubated with a rabbit anti-PTPIP51 and mouse anti-VAPB antibody for a PLA assay. Negative controls are shown in Fig. . Scale bar = 10 μm. ( G ) Quantification of the PLA assay (number of red dots per cell) shown in ( F ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p = 0.0016. ( H ) HEK293 cells expressing STIM1(full-length)-GFP were transfected for transient expression of a Flag-tagged STIM1 peptide encompassing residues 551-611. After 24 h of transfection, STIM1-GFP was immunoprecipitated and co-precipitated GRP75 was assessed by immunoblotting. Total levels of GRP75 and STIM1-GFP from WCL are shown in Fig. . ( I ) Quantification of co-precipitated GRP75 from 2 independent experiments and 3 technical replicates. Statistical analysis with unpaired t-test, p = 0.0072. Data are plotted as mean ± S.D. ( J ) HEK293 cells stably and inducibly expressing mito 4× -GCaMP6f were transiently transfected for the expression of mCherry-tagged STIM1 peptide corresponding to residues 551-611. The peptide was tagged with mCherry to select transfected cells, and the ratios F/Fr were calculated in mCherry-expressing cells only. The graph shows Fmax/Fr after the addition of ATP+CCh in Ca 2+ -free medium. Statistical analysis with unpaired t-test, p = 0.0407. Dots represent data from individual cells. ( K ) Analysis of the area under the curve (AUC) of the fluorescence profile after the ATP+CCh stimulus. A total of 16 independent experiments were performed in control (WT) cells ( n = 67 cells) and 17 experiments for cells expressing the peptide (551-611)-mCherry ( n = 44 cells). The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, p = 0.0021. ( L ) HEK293 cells inducibly expressing the Flag-tagged STIM1(551-611)-peptide or HEK293 control cells (with no peptide expression) were fixed and incubated with anti-VAPB and anti-PTPIP51 antibodies and PLA reagents, as in ( F ). Scale bar = 10 μm. ( M ) Quantification of VAPB-PTPIP51 interactions per cell from ( L ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p < 0.0001. ( N ) Wild-type HEK293 cells inducibly expressing the Flag-STIM1(551-611) peptide (red line) and loaded with fura-2 were assessed for the extension of SOCE and compared to cells with no peptide expression (black line). A total number of 51 (WT) and 49 (WT + peptide) cells were analyzed from 3 independent experiments. Data are plotted as mean ± S.D. .

Article Snippet: Rabbit anti-GRP75 (1:1000 in TBS-T 0.1% + 5% BSA, for IB. 1:100 in blocking buffer, for IF) , Cell Signaling Technology , 3593.

Techniques: Generated, Immunoprecipitation, Expressing, Mutagenesis, Plasmid Preparation, Control, Western Blot, Positive Control, Incubation, Transfection, Stable Transfection, Fluorescence

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Schematic representation of STIM1 mutants with deletions in the 551-685 region. The specific sequences deleted between the amino acids 551 and 685 for each of the generated STIM1 mutants are shown. This diagram was created with BioRender.com. ( B ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ643-677)-GFP (mutant 7), STIM1(Δ668-674)-GFP (mutant 8), STIM1(Δ672-685)-GFP (mutant 9), or GFP (empty vector) as a control. Co-precipitation of GRP75 was assessed by immunoblotting. WCL (3 µg) from cells expressing STIM1-GFP was loaded as a positive control. Total immunoprecipitated GFP was used as a loading control. Total levels of STIM1 and GRP75 in WCL (30 μg protein/lane) were analyzed by immunoblotting (Fig. ). ( C ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ551-611)-GFP (mutant 5), STIM1(Δ582-642)-GFP (mutant 6), STIM1(Δ672-685)-GFP (mutant 9), or GFP only as a control. Other conditions as stated for ( B ). Total levels of STIM1 and GRP75 in WCL were analyzed by immunoblotting (30 μg protein/lane) (Fig. ). ( D ) STIM1-KO HEK293 cells inducibly expressing full-length (FL) STIM1-GFP or STIM1(Δ551-611)-GFP were fixed and incubated with rabbit anti-GRP75 and/or sheep anti-GFP antibodies. Negative controls consisted of cells incubated with single primary antibodies. Scale bar = 10 μm. ( E ) Quantification of the interactions per cell detected by PLA in ( D ). The number of cells evaluated is indicated in parentheses, and the mean is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001 and p = 0.5354 (n.s.). ( F ) STIM1-KO HEK293 cells expressing STIM1-GFP (full-length, FL) or STIM1(Δ551-611)-GFP were incubated with a rabbit anti-PTPIP51 and mouse anti-VAPB antibody for a PLA assay. Negative controls are shown in Fig. . Scale bar = 10 μm. ( G ) Quantification of the PLA assay (number of red dots per cell) shown in ( F ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p = 0.0016. ( H ) HEK293 cells expressing STIM1(full-length)-GFP were transfected for transient expression of a Flag-tagged STIM1 peptide encompassing residues 551-611. After 24 h of transfection, STIM1-GFP was immunoprecipitated and co-precipitated GRP75 was assessed by immunoblotting. Total levels of GRP75 and STIM1-GFP from WCL are shown in Fig. . ( I ) Quantification of co-precipitated GRP75 from 2 independent experiments and 3 technical replicates. Statistical analysis with unpaired t-test, p = 0.0072. Data are plotted as mean ± S.D. ( J ) HEK293 cells stably and inducibly expressing mito 4× -GCaMP6f were transiently transfected for the expression of mCherry-tagged STIM1 peptide corresponding to residues 551-611. The peptide was tagged with mCherry to select transfected cells, and the ratios F/Fr were calculated in mCherry-expressing cells only. The graph shows Fmax/Fr after the addition of ATP+CCh in Ca 2+ -free medium. Statistical analysis with unpaired t-test, p = 0.0407. Dots represent data from individual cells. ( K ) Analysis of the area under the curve (AUC) of the fluorescence profile after the ATP+CCh stimulus. A total of 16 independent experiments were performed in control (WT) cells ( n = 67 cells) and 17 experiments for cells expressing the peptide (551-611)-mCherry ( n = 44 cells). The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, p = 0.0021. ( L ) HEK293 cells inducibly expressing the Flag-tagged STIM1(551-611)-peptide or HEK293 control cells (with no peptide expression) were fixed and incubated with anti-VAPB and anti-PTPIP51 antibodies and PLA reagents, as in ( F ). Scale bar = 10 μm. ( M ) Quantification of VAPB-PTPIP51 interactions per cell from ( L ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p < 0.0001. ( N ) Wild-type HEK293 cells inducibly expressing the Flag-STIM1(551-611) peptide (red line) and loaded with fura-2 were assessed for the extension of SOCE and compared to cells with no peptide expression (black line). A total number of 51 (WT) and 49 (WT + peptide) cells were analyzed from 3 independent experiments. Data are plotted as mean ± S.D. .

Article Snippet: Rabbit anti-GRP75 (1:600 in blocking buffer, for IF) , Proteintech , 14887-1-AP.

Techniques: Generated, Immunoprecipitation, Expressing, Mutagenesis, Plasmid Preparation, Control, Western Blot, Positive Control, Incubation, Transfection, Stable Transfection, Fluorescence

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: Identification of GRP75 as an HEV-ORF2-interacting protein. ( A ) Recombinant K239 protein forms virus-like particles (VLPs) resembling HEV capsids. Truncation of HEV-ORF2 forms VLP. The K239 protein was expressed in E. coli , refolded in PBS, and analyzed using electron microscopy (EM) to confirm VLP formation. Scale bar = 200 nm. ( B ). Identification of potential protein interactions with K239. The recombinant K239 protein was conjugated to CNBr-activated Sepharose 4B resin and incubated with plasma membrane extracts from S10-3 cells. SDS-PAGE and subsequent silver staining were performed to visualize potential interacting proteins. Plain CNBr-activated Sepharose 4B resin and recombinant SUMO protein-conjugated Sepharose 4B resin served as blank and irrelevant protein controls, respectively. ( C ) Western blot verification of GRP75 interaction with K239. K239-conjugated CNBr-activated Sepharose 4B resin was used to probe plasma membrane extracts from S10-3 cells. After SDS-PAGE, western blotting with an anti-GRP75 antibody was performed to confirm the presence of GRP75 in the pull-down complex. ( D ) HEV replication inhibits GRP75 expression in S10-3 cells. S10-3 cells were transfected with HEV-RNA (KernowC1-p6 strain), and samples were collected at various time points. Western blotting was performed to assess GRP75 protein levels over time. ( E ) Quantification of GRP75 mRNA in HEV-replicating S10-3 cells. Total RNA was extracted from S10-3 cells at different time points post-transfection using TRIzol reagent. The qPCR was performed to measure GRP75 mRNA expression, using GAPDH mRNA as an internal control. GRP75 mRNA levels from uninfected S10-3 cells collected at the same time points were included as controls. Data are presented as mean ± SD and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. All results are based on at least three independent biological replicates. ( F ) Quantification of HEV RNA in HEV-replicating S10-3 cells. Total RNA was extracted from S10-3 cells at different time points post-transfection using TRIzol reagent. Then, qPCR was performed to evaluate replication of HEV-RNA. Data are presented as mean ± SD and analyzed using Student’s t -test. ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Recombinant, Virus, Electron Microscopy, Incubation, Clinical Proteomics, Membrane, SDS Page, Silver Staining, Western Blot, Expressing, Transfection, Control

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75 interacts with HEV-ORF2 through its substrate-binding domain. ( A ) SDS-PAGE analysis for recombinant GRP75. Recombinant GRP75 was expressed from the E. coli system and refolded into PBS, then subjected to SDS-PAGE analysis. ( B ) Far-western blot assay for detecting the interaction between K239 and GRP75. Different doses of recombinant K239 protein were subjected to SDS-PAGE and transferred to a PVDF membrane. Next, the membrane was incubated with recombinant GRP75 protein expressed in E. coli , followed by detection using an anti-GRP75 antibody and corresponding secondary antibody to assess the binding between K239 and recombinant GRP75. Recombinant SUMO protein was included as an irrelevant control. ( C ) Co-immunoprecipitation (CoIP) analysis of the GRP75 and HEV-ORF2 interaction in HEV-infected HepG2/C3A cells. HepG2/C3A cells stably infected with HEV-3 KernowC1-p6 were lysed using NP-40 buffer and subjected to immunoprecipitation (IP) with an ORF2-specific monoclonal antibody (Mab) 2G8 to detect GRP75 within the ORF2 complex using a GRP75-specific antibody. Normal mouse IgG (mIgG) served as an antibody isotype control for IP. ( D ) CoIP assay for GRP75 and HEV-ORF2 interaction in HEV RNA-transfected S10-3 cells. S10-3 cells were transfected with HEV-3 KernowC1-p6 RNA and cultured for 7 days. Cells were then harvested for Co-IP analysis, following the same protocol as described above. Uninfected control cells were included for comparison. ( E ) Schematic illustration of GRP75 function domains. ( F ) The interaction of GRP75 with ORF2 depends on the substrate-binding domain (SBD) of GRP75. HEK-293T cells were transfected with plasmids encoding MYC-tagged full-length GRP75 (GRP75-MYC), nucleotide-binding domain (NBD) (NBD-MYC), or substrate binding domain (SBD-MYC), along with a plasmid encoding HEV-ORF2, for 48 h. Next, cells were lysed with NP-40 buffer, and proteins were immunoprecipitated using the ORF2-specific Mab-2G8, followed by western blotting using a MYC tag-specific monoclonal antibody to elucidate domain-specific interactions. The proteins from whole cell lysate (WCL) were probed to confirm the expression of the transfected plasmids.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Binding Assay, SDS Page, Recombinant, Far Western Blot, Membrane, Incubation, Control, Immunoprecipitation, Infection, Stable Transfection, Co-Immunoprecipitation Assay, Transfection, Cell Culture, Comparison, Plasmid Preparation, Western Blot, Expressing