vdac1 monoclonal antibody  (Proteintech)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: LINC01013 interacts with HSPA9. A ANKHD1, HSPA9 and DDX24 were identified as the candidate target proteins by catRAPID ( http://s.tartaglialab.com/page/catrapid_group_old ) and mass spectrometry (MS). B The heatmap results showed that LINC01013 can interact with the HSPA9 protein. C Schrodinger2019.01 software predicted and visualized the 3-dimensional structural docking of LINC01013 and HSPA9. D RNA pull-down followed by western blotting assays were used to detect the interaction between LINC01013 and the HSPA9 protein. E RNA immunoprecipitation (RIP) experiments demonstrated the association of HSPA9 with LINC01013 ( n = 4). F RNA-FISH and immunofluorescence assay were used to observe the colocalization of LINC01013 (red) and HSPA9 protein (green). The Pearson correlation coefficient obtained through ImageJ reflects the correlation between the 2 fluorescence labels in colocalization, a non-targeting FISH probe was used as a negative control (NC). Scale bar, 50 μm. All values are presented as the mean ± SEM. Statistical analysis was performed with Student’s t-test. * p < 0.05. NC, negative control; IP, immunoprecipitation; IgG, Immunoglobulin G

Article Snippet: Then, the cells were incubated with antibodies against HSPA9 (14887-1-AP, 1:100, Proteintech, IL, USA) at 4 °C overnight.

Techniques: Mass Spectrometry, Software, Western Blot, RNA Immunoprecipitation, Immunofluorescence, Fluorescence, Negative Control, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: LINC01013 disrupts mitochondrial homeostasis by scaffolding the RNA-binding protein HSPA9-mediated VDAC1 oligomerization under hypoxia exposure. A Proteins possible interacting with HSPA9 were predicted from STRING database ( https://cn.string-db.org/ ) (left). Coimmunoprecipitation (Co-IP) assay confirmed that LINC01013 knockdown affected the binding of HSPA9 and VDAC1 (right). B Schrodinger2019.01 software predicted and visualized the 3-dimensional structural docking of VDAC1 and HSPA9. C Effect of EGS on monomer and oligomers of VDAC1 in hPASMCs. The crosslinking reagent EGS was used to stabilize the oligomers during electrophoresis ( n = 5) D Western blotting of VDAC1 monomer and oligomers in hPASMCs ( n = 5). E Overexpression efficiency of HSPA9 were quantified by Western blotting ( n = 6). F Effects of LINC01013 and HSPA9 on VDAC1 oligomerization. G Representative images showing mitochondrial reactive oxygen species (MitoSOX, red) staining in hPASMCs ( n = 5). Scale bars, 50 μm. H Immunostaining of JC-1 aggregates (red) and monomers (green) showing changes in MMP across different groups. CCCP (10µM) was used as a positive control to induce MMP loss ( n = 5). Scale bars, 50 μm. All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA; oe, overexpression; MFI, Mean Fluorescence Intensity; CCCP, Carbonyl Cyanide 3-Chlorophenylhydrazone

Article Snippet: Then, the cells were incubated with antibodies against HSPA9 (14887-1-AP, 1:100, Proteintech, IL, USA) at 4 °C overnight.

Techniques: Scaffolding, RNA Binding Assay, Co-Immunoprecipitation Assay, Knockdown, Binding Assay, Software, Electrophoresis, Western Blot, Over Expression, Staining, Immunostaining, Positive Control, Negative Control, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Human LINC01013 delivery aggravates PH progression in mice. A - B Right ventricular systolic pressure (RVSP) and RV/left ventricular (LV) + Septum weight ratio in the SuHx-induced PH mouse models ( n = 6). C Pulmonary artery velocity time integral (PAVTI), pulmonary artery acceleration time (PAAT) and left ventricular ejection fraction (LVEF) of the SuHx-induced PH mice models infected with AAV5 carrying human LINC01013 ( n = 6). D Expression of PCNA in lung tissues ( n = 5). E Pulmonary arterial morphological analysis was performed by using hematoxylin and eosin (HE) and Masson staining. Scale bar, 100 μm. F-G Immunofluorescence of TNF-α, and activities of SOD and Gpx in lung tissues ( n = 6). Scale bars, 50 μm. H RNA pull-down assays detected the interaction between LINC01013 and HSPA9 protein in mouse PASMCs. All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; SuHx, hypoxic + Su5416; NC, negative control; ▲SMC, smooth muscle cell targeting

Article Snippet: Then, the cells were incubated with antibodies against HSPA9 (14887-1-AP, 1:100, Proteintech, IL, USA) at 4 °C overnight.

Techniques: Infection, Expressing, Staining, Immunofluorescence, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: A schematic diagram illustrating the hypothetical role of LINC01013 in hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) fate. In the nucleus, Ttranscription factor CEBPB directly bound SE and promoter region of LINC01013, and activated the transcription of LINC01013 in hypoxic PASMCs. In the mitochondria, LINC01013 as a protein-binding scaffold connecting HSPA9 and VDAC1, contributing to the oligomerization of VDAC1 and mitochondrial dysfunction by including oxidative stress and metabolic disorders, and ultimately leading to the proliferation and inflammation of PASMCs. SE, super-enhancer; ROS, reactive oxygen species; PASMCs, pulmonary arterial smooth muscle cells; HSPA9, heat shock protein family A (Hsp70) member 9; VDAC1, voltage dependent anion channel 1; CEBPB, CCAAT enhancer binding protein beta; SOD, superoxide dismutase; GPx, glutathione peroxidase; HK II, hexokinase 2; PKM2, pyruvate kinase M 2; PDH, pyruvate dehydrogenase

Article Snippet: Then, the cells were incubated with antibodies against HSPA9 (14887-1-AP, 1:100, Proteintech, IL, USA) at 4 °C overnight.

Techniques: Protein Binding, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: LINC01013 interacts with HSPA9. A ANKHD1, HSPA9 and DDX24 were identified as the candidate target proteins by catRAPID ( http://s.tartaglialab.com/page/catrapid_group_old ) and mass spectrometry (MS). B The heatmap results showed that LINC01013 can interact with the HSPA9 protein. C Schrodinger2019.01 software predicted and visualized the 3-dimensional structural docking of LINC01013 and HSPA9. D RNA pull-down followed by western blotting assays were used to detect the interaction between LINC01013 and the HSPA9 protein. E RNA immunoprecipitation (RIP) experiments demonstrated the association of HSPA9 with LINC01013 ( n = 4). F RNA-FISH and immunofluorescence assay were used to observe the colocalization of LINC01013 (red) and HSPA9 protein (green). The Pearson correlation coefficient obtained through ImageJ reflects the correlation between the 2 fluorescence labels in colocalization, a non-targeting FISH probe was used as a negative control (NC). Scale bar, 50 μm. All values are presented as the mean ± SEM. Statistical analysis was performed with Student’s t-test. * p < 0.05. NC, negative control; IP, immunoprecipitation; IgG, Immunoglobulin G

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Mass Spectrometry, Software, Western Blot, RNA Immunoprecipitation, Immunofluorescence, Fluorescence, Negative Control, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: LINC01013 disrupts mitochondrial homeostasis by scaffolding the RNA-binding protein HSPA9-mediated VDAC1 oligomerization under hypoxia exposure. A Proteins possible interacting with HSPA9 were predicted from STRING database ( https://cn.string-db.org/ ) (left). Coimmunoprecipitation (Co-IP) assay confirmed that LINC01013 knockdown affected the binding of HSPA9 and VDAC1 (right). B Schrodinger2019.01 software predicted and visualized the 3-dimensional structural docking of VDAC1 and HSPA9. C Effect of EGS on monomer and oligomers of VDAC1 in hPASMCs. The crosslinking reagent EGS was used to stabilize the oligomers during electrophoresis ( n = 5) D Western blotting of VDAC1 monomer and oligomers in hPASMCs ( n = 5). E Overexpression efficiency of HSPA9 were quantified by Western blotting ( n = 6). F Effects of LINC01013 and HSPA9 on VDAC1 oligomerization. G Representative images showing mitochondrial reactive oxygen species (MitoSOX, red) staining in hPASMCs ( n = 5). Scale bars, 50 μm. H Immunostaining of JC-1 aggregates (red) and monomers (green) showing changes in MMP across different groups. CCCP (10µM) was used as a positive control to induce MMP loss ( n = 5). Scale bars, 50 μm. All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA; oe, overexpression; MFI, Mean Fluorescence Intensity; CCCP, Carbonyl Cyanide 3-Chlorophenylhydrazone

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Scaffolding, RNA Binding Assay, Co-Immunoprecipitation Assay, Knockdown, Binding Assay, Software, Electrophoresis, Western Blot, Over Expression, Staining, Immunostaining, Positive Control, Negative Control, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Human LINC01013 delivery aggravates PH progression in mice. A - B Right ventricular systolic pressure (RVSP) and RV/left ventricular (LV) + Septum weight ratio in the SuHx-induced PH mouse models ( n = 6). C Pulmonary artery velocity time integral (PAVTI), pulmonary artery acceleration time (PAAT) and left ventricular ejection fraction (LVEF) of the SuHx-induced PH mice models infected with AAV5 carrying human LINC01013 ( n = 6). D Expression of PCNA in lung tissues ( n = 5). E Pulmonary arterial morphological analysis was performed by using hematoxylin and eosin (HE) and Masson staining. Scale bar, 100 μm. F-G Immunofluorescence of TNF-α, and activities of SOD and Gpx in lung tissues ( n = 6). Scale bars, 50 μm. H RNA pull-down assays detected the interaction between LINC01013 and HSPA9 protein in mouse PASMCs. All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; SuHx, hypoxic + Su5416; NC, negative control; ▲SMC, smooth muscle cell targeting

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Infection, Expressing, Staining, Immunofluorescence, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: A schematic diagram illustrating the hypothetical role of LINC01013 in hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) fate. In the nucleus, Ttranscription factor CEBPB directly bound SE and promoter region of LINC01013, and activated the transcription of LINC01013 in hypoxic PASMCs. In the mitochondria, LINC01013 as a protein-binding scaffold connecting HSPA9 and VDAC1, contributing to the oligomerization of VDAC1 and mitochondrial dysfunction by including oxidative stress and metabolic disorders, and ultimately leading to the proliferation and inflammation of PASMCs. SE, super-enhancer; ROS, reactive oxygen species; PASMCs, pulmonary arterial smooth muscle cells; HSPA9, heat shock protein family A (Hsp70) member 9; VDAC1, voltage dependent anion channel 1; CEBPB, CCAAT enhancer binding protein beta; SOD, superoxide dismutase; GPx, glutathione peroxidase; HK II, hexokinase 2; PKM2, pyruvate kinase M 2; PDH, pyruvate dehydrogenase

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Protein Binding, Binding Assay

Journal: Molecular Neurobiology

Article Title: Metformin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage Via Improving Endoplasmic Reticulum Stress and Mitochondrial Stress-Mediated Ca 2+ Imbalance

doi: 10.1007/s12035-025-05558-1

Figure Lengend Snippet: ERSand MS mediated the IP3R1–GRP75–VDAC1 Ca2 + channeling complex in the first 72 h following SAH in the temporal cortex of mice in vivo. A – D Representative Western blot band and densitometric quantification of the time-dependent expression of IP3R1, GRP75, and VDAC1 in the temporal cortex of SAH mice models in the initial 72 h. The expression of the IP3R1–GRP75–VDAC1 complex was upregulated. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Samples derived from the same experiment, and the gels were processed in parallel. Protein expression levels were normalized to GAPDH as an internal control. Individual gels were run for the separate proteins when the loading protein amount was consistent. E – F Conventional immunofluorescence of PTP1B, Calnexin, VDAC1, and DAPI in the bilateral temporal cortex and CA3. The subsequent MAM junction variation was detected by the conventional immunofluorescence colocalization of calnexin and VDAC1 in vivo. Scale bar = 50 μm, N = 4 per group. G – I The outline and the proportion of surviving neurons induced by SAH in the bilateral temporal cortex and CA3 region. Scale bar = 50 μm, N = 4 per group. J The TEM showed dilated rough ER fragments and swollen mitochondria in the bilateral temporal cortex in each group. Images were acquired at a magnification of 20,000 × , Scale bar = 250 nm. An unpaired Student’s t test was used for comparisons between two groups, whereas ANOVA followed by Tukey’s post hoc test was used for comparisons among multiple groups

Article Snippet: The PVDF membrane was blocked in 5% skim milk for 90 min at room temperature, followed by incubation with the primary antibodies against PTP1B (1:2000 Proteintech, catalog number 11334–1-AP), IP3R1 (1:2000, Abcam, catalog number ab264281), CHOP (1:1000, Proteintech, catalog number 15204–1-AP), Bcl-2 (1:2000, Affinity, lot# 70g9181), Bax (1:2000, Proteintech, catalog number 50599–2-2 g), VDAC1 (1:2000, Proteintech, catalog number 10866–1-AP), AKT (1:1000 Proteintech, catalog number 10176–2-AP), p-AKT (1:1000 Proteintech, catalog number 80455–1-RR), GRP75 (1:1000, Bio-Techne, catalog number MAB3584, ATF4 (1:2000, Proteintech, catalog number 10835–1-AP, p- eIF2a (1:500, Affinity, catalog number AF3087), eIF2a (1:1000, Proteintech, catalog number 11170–1-AP), and CC3 (1:1000, Bioss, Boston, MA, USA lot: BJ03319208), respectively.

Techniques: In Vivo, Western Blot, Expressing, Derivative Assay, Control, Immunofluorescence

Journal: Molecular Neurobiology

Article Title: Metformin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage Via Improving Endoplasmic Reticulum Stress and Mitochondrial Stress-Mediated Ca 2+ Imbalance

doi: 10.1007/s12035-025-05558-1

Figure Lengend Snippet: Met significantly suppressed Ca2 + transfer from the ER to the mitochondria through the PTP1B/AKT following SAH 24 h in the temporal cortex of mice in vivo. A – G To determine the regulatory role of Met in ER stress and mitochondrial signaling, we employed lentivirus-mediated modulation in a 24-h SAH model and confirmed the effects by Western blot analysis. Representative Western blot band and densitometric quantification of the time-dependent expression of CHOP, ATF4, PTP1B, p-eIF2a, eIF2a, p-AKT, and AKT following SAH 24 h in vivo. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Samples derived from the same experiment, and the gels were processed in parallel. Protein expression levels were normalized to GAPDH as an internal control. Individual gels were run for the separate proteins when the loading protein amount was consistent. H The effect of Met on the concentration of Ca 2+ in the temporal cortex of SAH groups. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). I – L Representative Western blot band and densitometric quantification of the time-dependent expression of IP3R1, GRP75, and VDAC1 in vivo. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). M , N The effect of Met on the conventional immunofluorescence of PTP1B, calnexin, VDAC1, and DAPI in the bilateral temporal cortex and CA3. The subsequent MAM junction variation was detected by the conventional immunofluorescence colocalization of calnexin and VDAC1 in vivo. Scale bar = 50 μm, N = 4 per group. An unpaired Student’s t test was used for comparisons between two groups, whereas ANOVA followed by Tukey’s post hoc test was used for comparisons among multiple groups

Article Snippet: The PVDF membrane was blocked in 5% skim milk for 90 min at room temperature, followed by incubation with the primary antibodies against PTP1B (1:2000 Proteintech, catalog number 11334–1-AP), IP3R1 (1:2000, Abcam, catalog number ab264281), CHOP (1:1000, Proteintech, catalog number 15204–1-AP), Bcl-2 (1:2000, Affinity, lot# 70g9181), Bax (1:2000, Proteintech, catalog number 50599–2-2 g), VDAC1 (1:2000, Proteintech, catalog number 10866–1-AP), AKT (1:1000 Proteintech, catalog number 10176–2-AP), p-AKT (1:1000 Proteintech, catalog number 80455–1-RR), GRP75 (1:1000, Bio-Techne, catalog number MAB3584, ATF4 (1:2000, Proteintech, catalog number 10835–1-AP, p- eIF2a (1:500, Affinity, catalog number AF3087), eIF2a (1:1000, Proteintech, catalog number 11170–1-AP), and CC3 (1:1000, Bioss, Boston, MA, USA lot: BJ03319208), respectively.

Techniques: In Vivo, Western Blot, Expressing, Derivative Assay, Control, Concentration Assay, Immunofluorescence

Journal: Molecular Neurobiology

Article Title: Metformin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage Via Improving Endoplasmic Reticulum Stress and Mitochondrial Stress-Mediated Ca 2+ Imbalance

doi: 10.1007/s12035-025-05558-1

Figure Lengend Snippet: Met significantly suppressed Ca 2+ transfer from the ER to the mitochondria through the PTP1B/AKT following SAH 24 h in vitro. A – K Our vitro data demonstrate that PTP1B plays a critical role in integrating ER stress with mitochondrial dysfunction following SAH 24 h. Representative Western blot band and densitometric quantification of PTP1B, p-AKT, AKT, p-eIF2a, eIF2a, IP3R1, GRP75, VDAC1, ATF4, and CHOP in primary neurons induced by the OxyHb. The expression of the IP3R1–GRP75–VDAC1 complex was upregulated accordingly. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Samples derived from the same experiment, and the gels were processed in parallel. Protein expression levels were normalized to GAPDH as an internal control. Individual gels were run for the separate proteins when the loading protein amount was consistent. L Conventional immunofluorescence confirmed the colocalization of NEUN, MAP-2, and DAPI in primary neurons. Scale bar = 50 μm. M – R The effect of Met on the apoptosis of SAH in vitro. Representative Western blot bands of Bcl-2, Bax, CC3, and flow cytometry were used to evaluate the effect of Met on apoptosis in vivo. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Protein expression levels were normalized to GAPDH as an internal control. S The conventional immunofluorescence staining showed that the colocalization of intracellular Ca 2+ concentration increased significantly in the OxyHb24H group in primary neurons. Scale bar = 50 μm. T The conventional immunofluorescence confirmed the colocalization of PTP1B, calnexin, VDAC1, and DAPI in primary neurons after the intervention of lentivirus and inhibitors. To assess the effect of Met on MAM integrity, we performed immunofluorescence co-localization analysis of the ER marker calnexin and the mitochondrial marker VDAC1 in treated primary neurons. Scale bar = 50 μm. An unpaired Student’s t test was used for comparisons between two groups, whereas ANOVA followed by Tukey’s post hoc test was used for comparisons among multiple groups

Article Snippet: The PVDF membrane was blocked in 5% skim milk for 90 min at room temperature, followed by incubation with the primary antibodies against PTP1B (1:2000 Proteintech, catalog number 11334–1-AP), IP3R1 (1:2000, Abcam, catalog number ab264281), CHOP (1:1000, Proteintech, catalog number 15204–1-AP), Bcl-2 (1:2000, Affinity, lot# 70g9181), Bax (1:2000, Proteintech, catalog number 50599–2-2 g), VDAC1 (1:2000, Proteintech, catalog number 10866–1-AP), AKT (1:1000 Proteintech, catalog number 10176–2-AP), p-AKT (1:1000 Proteintech, catalog number 80455–1-RR), GRP75 (1:1000, Bio-Techne, catalog number MAB3584, ATF4 (1:2000, Proteintech, catalog number 10835–1-AP, p- eIF2a (1:500, Affinity, catalog number AF3087), eIF2a (1:1000, Proteintech, catalog number 11170–1-AP), and CC3 (1:1000, Bioss, Boston, MA, USA lot: BJ03319208), respectively.

Techniques: In Vitro, Western Blot, Expressing, Derivative Assay, Control, Immunofluorescence, Flow Cytometry, In Vivo, Staining, Concentration Assay, Marker

Journal: Molecular Neurobiology

Article Title: Metformin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage Via Improving Endoplasmic Reticulum Stress and Mitochondrial Stress-Mediated Ca 2+ Imbalance

doi: 10.1007/s12035-025-05558-1

Figure Lengend Snippet: Metformin ameliorates early brain injury after subarachnoid hemorrhage via improving ERS and MS-mediated Ca 2+ imbalance. The IP3R1–GRP75–VDAC1 complex regulates MAM and plays an imperative role in EBI. The treatment of Met ameliorates ER stress, MS, and Ca 2+ overload, further improving neuroapoptosis and alleviating the EBI of SAH

Article Snippet: The PVDF membrane was blocked in 5% skim milk for 90 min at room temperature, followed by incubation with the primary antibodies against PTP1B (1:2000 Proteintech, catalog number 11334–1-AP), IP3R1 (1:2000, Abcam, catalog number ab264281), CHOP (1:1000, Proteintech, catalog number 15204–1-AP), Bcl-2 (1:2000, Affinity, lot# 70g9181), Bax (1:2000, Proteintech, catalog number 50599–2-2 g), VDAC1 (1:2000, Proteintech, catalog number 10866–1-AP), AKT (1:1000 Proteintech, catalog number 10176–2-AP), p-AKT (1:1000 Proteintech, catalog number 80455–1-RR), GRP75 (1:1000, Bio-Techne, catalog number MAB3584, ATF4 (1:2000, Proteintech, catalog number 10835–1-AP, p- eIF2a (1:500, Affinity, catalog number AF3087), eIF2a (1:1000, Proteintech, catalog number 11170–1-AP), and CC3 (1:1000, Bioss, Boston, MA, USA lot: BJ03319208), respectively.

Techniques: