anti hspa4 antibody  (Proteintech)

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A Overexpression of HSPA4 in SH-SY5Y cells. The cells were transfected with a plasmid harboring Hspa4 for 24 h. The protein levels of HSPA4 were analyzed by western blot, and Actin was used as a loading control. n = 3 independent experiments. B Cell morphology showing that HSPA4 protects cells from erastin-induced insults. SH-SY5Y cells were transfected with the plasmid expressing Hspa4 for 24 h, after which the cells were exposed to 5 μM erastin for an additional 24 h. Cell images were taken with a microscope. Scale bar = 50 μm. C HSPA4 increases the viability of erastin-treated cells. The cell treatments used are described in ( B ). Cell viability was assayed by the method of MTT. n = 8 biologically independent samples. D The expression of HSPA4 was increased by transfection with a plasmid carrying Hspa4 . The cell treatments are described in ( B ). The protein levels of HSPA4 were analyzed by western blot, and Actin was used as a loading control. n = 3 independent experiments. E HSPA4 reduces LDH release in erastin-treated cells. The cell treatments are described in ( B ). n = 4 biologically independent samples. F JC-1 staining showing that HSPA4 increases the mitochondrial membrane potential in erastin-treated cells. The cell treatments are described in ( B ). Scale bar = 50 μm. n = 5 biologically independent samples. G HSPA4 decreases mitochondrial oxidative stress in erastin-treated cells. SH-SY5Y cells were co-transfected with the plasmids expressing Hspa4 or pMito Timer. Twenty-four hours post-transfection, the cells were treated with 5 μM erastin for an additional 24 h. Scale bar = 25 μm. n = 5 biologically independent samples. Data represent means ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Control, Expressing, Microscopy, Staining, Membrane

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A HSPA4 reduces ROS production in erastin-treated cells. SH-SY5Y cells were transfected with a plasmid expressing Hspa4 for 24 h, after which the cells were exposed to 5 μM erastin for an additional 24 h. Intracellular ROS generation was measured using a BODIPY (581/591) C11 probe. O-BODIPY: oxidized BODIPY; R-BODIPY: reduced BODIPY. Scale bar = 50 μm. B The quantified fluorescence intensity based on the images shown in ( A ). n = 3 biologically independent samples. C HSPA4 increases the level of intracellular GSH in erastin-treated cells. The cell treatments are described in ( A ). n = 3 biologically independent samples. D , E HSPA4 reduces intracellular MDA (D) and Fe 2+ (E) levels in erastin-treated cells. The cell treatments are described in ( A ). n = 3 biologically independent samples. F The protein levels of GPX4, FTH1 and xCT were increased by HSPA4 in erastin-treated cells. The cell treatments are described in ( A ). Protein expression was analyzed by western blot, and Actin was used as a loading control. n = 3 independent experiments. Data represent means ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Transfection, Plasmid Preparation, Expressing, Fluorescence, Western Blot, Control

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A , B HSPA4 expression was repressed by Hspa4 siRNAs in SH-SY5Y cells. The cells were transfected with three pairs of siRNAs against Hspa4 . Forty-eight hours post-transfection, HSPA4 expression was analyzed by qRT-PCR ( A ) or western blot ( B ). 18S rRNA was used a housekeeping gene for qRT-PCR, while Actin was used as a loading control for western blot. n = 3 biologically independent samples ( A ) or independent experiments ( B ). C Cell morphology showing that HSPA4 knockdown worsens the cell insults induced by erastin. SH-SY5Y cells were transfected with Hspa4 siRNA-1, and 48 h post-transfection, the cells were treated with 5 μM erastin for an additional 24 h. Cell images were taken with a microscope. Scale bar = 50 μm. D HSPA4 knockdown further decreased the viability of erastin-treated cells. The cell treatments are described in ( C ). n = 8 biologically independent samples. E LDH release was further increased by HSPA4 knockdown in erastin-treated cells. The cell treatments are described in ( C ). n = 4 biologically independent samples. F Mitochondrial membrane potential was worsened by HSPA4 knockdown. The cell treatments are described in ( C ). Scale bar = 50 μm. n = 4 biologically independent samples. Data represent means ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Control, Knockdown, Microscopy, Membrane

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A HSPA4 knockdown stimulates ROS production in erastin-treated cells. SH-SY5Y cells were transfected with Hspa4 siRNA-1. Forty-eight hours post-transfection, the cells were treated with 5 μM erastin for an additional 24 h. Intracellular ROS generation was measured by using a BODIPY (581/591) C11 probe. O-BODIPY: oxidized BODIPY; R-BODIPY: reduced BODIPY. Scale bar = 50 μm. B Fluorescence intensity quantified from the images shown in ( A ). n = 3 biologically independent samples. C Intracellular GSH was reduced by HSPA4 knockdown in erastin-treated cells. The cell treatments are described in ( A ). n = 3 biologically independent samples. D , E Intracellular MDA ( D ) and Fe 2+ ( E ) levels were increased by HSPA4 knockdown in erastin-treated cells. The cell treatments are described in ( A ). n = 3 biologically independent samples. F The protein levels of GPX4, FTH1, and xCT were further decreased by HSPA4 knockdown in erastin-treated cells. The cell treatments are described in ( A ). n = 3 independent experiments. Data represent means ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Knockdown, Transfection, Fluorescence

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A Experimental timeline. After behavioral training, the mice received AAV-HSPA4 via stereotaxic injection. Twenty-eight days post-injection, the mice were intraperitoneally administrated MPTP at 20 mg/kg/day for 7 days, after which they were subjected to behavioral tests. At the end of the experiments, the mice were sacrificed, and brain samples were collected for further analyses. B , C HSPA4 expression in the SNpc was increased by AAV-HSPA4. Immunofluorescence ( B ) or western blot ( C ) was used to examine HSPA4 expression. DAPI was used to stain the nucleus. Actin was used as a loading control. Scale bar = 100 μm. n = 3 biologically independent animals. D , E , F , G HSPA4 improves the behavioral performance in the rotarod test ( D ), pole test ( E ), traction test ( F ), and olfactory test ( G ). n = 6 biologically independent animals. Data represent means ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Injection, Expressing, Immunofluorescence, Western Blot, Staining, Control

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A The protein levels of GPX4, xCT, and FTH1 were increased by HSPA4 in the SNpc of PD model mice. Protein expression was analyzed by western blot, and Actin was used as a loading control. n = 3 independent experiments. B , C Perls’ Prussian blue staining showing that ion deposition was reduced by HSPA4 in the SNpc ( B ) and striatum ( C ) of PD model mice. n = 3 biologically independent animals. Scale bar = 200 μm. D HSPA4 improves mitochondrial integrity in the SNpc. Images of the mitochondria were obtained with an electron microscope. The white arrowheads indicate disrupted mitochondria. Scale bar = 1 μm. Data represent means ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Expressing, Western Blot, Control, Staining, Microscopy

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A Immunofluorescence analysis of Iba-1 expression in the SNpc. Scale bar = 200 μm. B Immunofluorescence analysis of Iba-1 expression in the striatum. Scale bar = 200 μm. C Immunofluorescence analysis of GFAP expression in the SNpc. Scale bar = 200 μm. D Immunofluorescence analysis of GFAP expression in the striatum. Scale bar = 200 μm. E The expression of Il1b , Il6 , and Il8 were reduced by HSPA4 in the SNpc and striatum of PD model mice. Gene expression was analyzed by qRT-PCR with 18S rRNA as an internal control. F The levels of IL-1β, IL-6, and IL-8 were decreased by HSPA4 in the SNpc and striatum of PD model mice. STR: striatum; Data represent means ± SEM. n = 5 biologically independent animals. Statistical significance was determined by one-way ANOVA with Tukey’s test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Immunofluorescence, Expressing, Gene Expression, Quantitative RT-PCR, Control

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A Identification of transferrin as a substrate of HSPA4. SH-SY5Y cells were transfected with a plasmid expressing SP-tagged Hspa4 . Twenty-four hours post-transfection, the cells were harvested for protein pull-down. The resulting samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). B Protein-protein docking analysis showing that HSPA4 (blue) has high affinity for transferrin (green). The enlarged part showing that HSPA4 interacts with transferrin via hydrogen bonds between specific amino acids. C HSPA4 interacts with transferrin. SH-SY5Y cells were transfected with a plasmid expressing SP-tagged Hspa4 . Twenty-four hours post-transfection, the cells were harvested and subjected to HSPA4 pull-down. The resulting pull-down samples were separated by SDS-PAGE and then subjected to Coomassie blue staining (left panel) and western blot analysis for HSPA4 (middle panel) or transferrin (right panel). M: marker; EV: empty vector; IB: immunoblot. D HSPA4 interacts with transferrin. SH-SY5Y cells were transfected with a plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cells were harvested and subjected to co-immunoprecipitation (co-IP) using an anti-HSPA4 antibody. The resulting co-IP samples and total cell lysates (input) were analyzed by western blot. Actin was used as a loading control.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Transfection, Plasmid Preparation, Expressing, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, SDS Page, Staining, Western Blot, Marker, Immunoprecipitation, Co-Immunoprecipitation Assay, Control

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A HSPA4 colocalizes with transferrin in cells. The expression of HSPA4 (green) and transferrin (TF; red) in SH-SY5Y cells was analyzed by immunofluorescence analysis, and DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. B Intracellular transferrin was increased by HSPA4. SH-SY5Y cells were transfected with the plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cells were subjected to immunofluorescence analysis using an anti-transferrin antibody. DAPI was used to stain the nucleus. Scale bar = 50 μm. C Quantification of the relative fluorescence intensity of transferrin, as shown in ( B ). n = 5 biologically independent samples. D HSPA4 sequesters transferrin in SH-SY5Y cells. SH-SY5Y cells were transfected with a plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cells and cell culture media were collected, and HSPA4 expression was analyzed by western blot. Actin (left panel) and Coomassie blue staining (right panel) were used as loading controls. E HSPA4 reduces extracellular transferrin. SH-SY5Y cells were transfected with a plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cell culture media were collected, and transferrin was measured with an ELISA kit. n = 4 biologically independent samples. F HSPA4 decreases the level of intracellular Fe 2+ . SH-SY5Y cells were transfected with a plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cells were harvested for analysis of Fe 2+ . n = 3 biologically independent samples. EV: empty vector; TF: transferrin. All data are presented as means ± SEM. Statistical significance was determined by unpaired Student’s t -test.

Article Snippet: Then, the cells were incubated with primary antibodies, including an anti-HSPA4 antibody (1:500; 21206-1-AP; Proteintech), an anti-transferrin antibody (1:200; 17435-1-AP; Proteintech), and an anti-transferrin receptor antibody (1:200; #13113; Cell Signaling Technology) overnight at 4 °C.

Techniques: Expressing, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Fluorescence, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: Identification of liver proteins as biomarker for postmortem diagnosis of heat stroke through proteomics.

doi: 10.1038/s41598-025-00963-x

Figure Lengend Snippet: Fig. 6. Three candidate protein biomarkers (HSPA4, GGCX, and CYP2B6) were significantly different between HS human cases and control cases. (a) Representative pictures of IHC staining from three candidate biomarkers (HSPA4, GGCX, and CYP2B6) in death from HS group and control group. (200 ×, Bar: 100 μm; 400 ×, Bar: 50 μm). (b) Statistical analysis of mean density values for the IHC staining between HS group and control group (***: P ≤ 0.001; ****: P ≤ 0.0005).

Article Snippet: The PVDF membranes were incubated in 5% skim milk for 2 h and then incubated in diluted primary antibody RASA4 (1:1500, PAB43047), SERPINA3 C (1:1000, 12,192–1- AP), HSPA4 (1:1000, 21,206–1-AP), GGCX (1:1000, PAB33008), CYP2B6 (1:1000, PAB32079), ACTB (1:2000, BM-3873, Boster), and GAPDH (1:120,00, 10,494–1-AP, Proteintech) at 4 °C overnight.

Techniques: Control, Immunohistochemistry

Journal: Scientific reports

Article Title: Identification of liver proteins as biomarker for postmortem diagnosis of heat stroke through proteomics.

doi: 10.1038/s41598-025-00963-x

Figure Lengend Snippet: Fig. 7. ROC curve analysis results of the diagnostic efficiency for HSPA4, GGCX, CYP2B6, and combination of the two proteins. AUC: Area Under the Curve.

Article Snippet: The PVDF membranes were incubated in 5% skim milk for 2 h and then incubated in diluted primary antibody RASA4 (1:1500, PAB43047), SERPINA3 C (1:1000, 12,192–1- AP), HSPA4 (1:1000, 21,206–1-AP), GGCX (1:1000, PAB33008), CYP2B6 (1:1000, PAB32079), ACTB (1:2000, BM-3873, Boster), and GAPDH (1:120,00, 10,494–1-AP, Proteintech) at 4 °C overnight.

Techniques: Diagnostic Assay