primary antibodies against mouse hsp 60  (Developmental Studies Hybridoma Bank)

Journal: Experimental Physiology

Article Title: Effect of prolonged voluntary wheel running on oxidative stress and defence mechanisms in cortex and hippocampus of healthy female rats

doi: 10.1113/ep092815

Figure Lengend Snippet: FIGURE 6 Mitochondrial BER. (a) Representative immunoblots of target proteins in mitochondrial extracts from cortex and hippocampus. Proteins probed for are shown on the left. APE1, apurinic/apyrimidinic endonuclease 1; HSP60, heat shock protein 60. (b) Representative gel from in vitro AP endonuclease activity assay with mitochondrial extracts from hippocampus. S, substrate; P, product; AP, oligonucleotide with AP site; C, control oligonucleotide without a lesion. (c–f) All values are normalized to HSP60 (mitochondrial loading control) and set relative to the sedentary group mean. Bars represent means ± SD. Differences were evaluated by Student’s unpaired t-test (c) or Mann–Whitney U-test (d–f). (c) n = 10 and 8 for sedentary and running group, respectively. (d) n = 10 and 7 for sedentary and running group, respectively. (e) n = 9 and 8 for sedentary and running group, respectively. (f) n = 7 and 9 for sedentary and running group, respectively. One outlier was excluded from the analysis in the sedentary group for (e, f). Other discrepancies in sample size are due to technical issues in assays or insufficient yield from mitochondrial purification. BER, base excision repair.

Article Snippet: anti-8-oxoguanine DNA glycosylase 1 (OGG1) (Novus Biologicals, cat. no. NB100-106, 1:500, RRID: AB_10104097), mouse anti-superoxide dismutase 1 (SOD1) (Santa Cruz Biotechnology, Dallas, TX, USA, cat. no. sc-101523, 1:500, RRID: AB_2191632), mouse anti-superoxide dismutase 2 (SOD2) (Santa Cruz Biotechnology, cat. no. sc-137254, 1:200, RRID: AB_2191808), mouse anti-catalase (CAT) (Santa Cruz Biotechnology, cat. no. sc-271803, 1:500, RRID: AB_10708550), mouse anti-heat shock protein 60 (HSP60) (Santa Cruz Biotechnology, cat. no. sc-271215, 1:1000, RRID: AB_10607973), and mouse antiβ-actin (Sigma-Aldrich, St Louis, MO, USA, cat. no. A2228, 1:10,000, RRID: 476697).

Techniques: Western Blot, In Vitro, Activity Assay, Control, MANN-WHITNEY, Purification

Journal: PLOS Pathogens

Article Title: Chlamydial protease-like activity factor targets SLC7A11 for degradation to induce ferroptosis and facilitate progeny releases

doi: 10.1371/journal.ppat.1013060

Figure Lengend Snippet: ( A ) A schematic diagram illustrates a series of reported pharmaceuticals that block ferroptosis by inhibiting lipid ROS accumulation. ( B ) The release of LDH and the levels of lipid ROS in Chlamydia trachomatis serovar D (MOI 10)-infected HeLa-229 cells were assessed following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. Statistical analysis was conducted using a one-way ANOVA with Bonferroni’s multiple comparisons (n=3). ( C ) Immunoblot analysis of chlamydial MOMP from cell supernatant and GAPDH from cell lysate were conducted following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. ( D ) The copy number of the chlamydial cryptic plasmid in the cell supernatant of Chlamydia trachomatis serovar D (MOI 10)-infected cells was determined following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. Statistical analysis was conducted using a one-way ANOVA with Bonferroni’s multiple comparisons (n=3). ( E ) The copy number of ompA in the total culture (cell supernatant and monolayer) of Chlamydia trachomatis serovar D (MOI 10)-infected cells was determined following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) over a time course. Statistical analysis was conducted using a two-way ANOVA test (n=3). ( F , G ) The release of LDH, lipid ROS levels, and the copy number of the chlamydial cryptic plasmid of cell supernatant in Chlamydia trachomatis serovars L1 (MOI 3)- ( F ) and A (MOI 3)- ( G ) infected cells were measured following treatment with liproxstatin-1 (1 μM) for 72 hours. The Student’s t - test was used for statistical analysis of ( F ) and ( G ) (n=3). ( H ) The release of LDH and the level of lipid ROS in Chlamydia muridarum (CM) (MOI 2)-infected McCoy cells were measured following treatment with trolox (3.2 mM) for 48 hours. The Student’s t - test was used for statistical analysis (n=3). ( I ) Immunoblot analysis of chlamydial HSP60 in the cell supernatant and β-actin in the cell lysate was performed following treatment with trolox (3.2 mM) for 48 hours. ( J ) The copy number of the Nigg II plasmid in the cell supernatant and total culture (cell supernatant and monolayer) of CM (MOI 2)-infected McCoy cells was quantified over a time-course following treatment with trolox (3.2 mM). The Student’s t-test was used for statistical analysis of the cell supernatant data (left panel), while a two-way ANOVA was applied to the data from the total culture (cell supernatant and monolayer) over the time course (right panel). Data are presented as the mean ± SD (n=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: For monitor the growth dynamic of CT, a mouse anti-chlamydial Hsp60 antibody conjugated to Alexa Fluor 488 (Santa Cruz; sc-57840 AF488) was used.

Techniques: Blocking Assay, Infection, Western Blot, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Chlamydial protease-like activity factor targets SLC7A11 for degradation to induce ferroptosis and facilitate progeny releases

doi: 10.1371/journal.ppat.1013060

Figure Lengend Snippet: (A) Immunoblot analysis of multiple ferroptosis-associated proteins and chlamydial HSP60 was performed in cells infected with various MOIs of Chlamydia trachomatis serovar D (CT-D) at 72 h.p.i., compared to mock-infected cells. (B) Immunoblot analysis of SLC7A11, GPx4, GAPDH, and chlamydial HSP60 was conducted in cells infected with Chlamydia trachomatis serovar L1 (CT-L1) (MOI 3) and Chlamydia muridarum (CM) (MOI 2), compared to mock-infected cells. (C) A schematic representation of the SLC7A11-GSH-GPx4 pathway in the regulation of ferroptosis. (D) Intracellular glutathione (GSH) levels were measured in CT-D (MOI 5)-infected cells at 72 h.p.i by flow cytometry, compared to mock-infected cells. The fraction of cells with high intracellular GSH was calculated. The Student’s t-test was used for statistical analysis. Data are presented as the mean ± SD (n=3). **, P < 0.01.

Article Snippet: For monitor the growth dynamic of CT, a mouse anti-chlamydial Hsp60 antibody conjugated to Alexa Fluor 488 (Santa Cruz; sc-57840 AF488) was used.

Techniques: Western Blot, Infection, Flow Cytometry

Journal: Scientific Reports

Article Title: Loss of O 6 -methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

doi: 10.1038/s41598-024-78885-3

Figure Lengend Snippet: Poly (I:C) stimulation increases the MGMT level in the nuclei and mitochondria of BMMs. ( A ) WT BMMs were stimulated with poly (I:C) (20 µg/ml) and incubated for 24 h. The cells were then fixed and stained for MGMT, HSP60 (a mitochondrial marker) and the nucleus. The cells were processed for confocal microscopy analysis. Scale bar = 5 µM. ( B ) MGMT puncta were quantified in at least 50 cells per condition per independent experiment. The data are presented as the means ± SEMs from three independent experiments. * P < 0.05. ( C ) Colocalization of MGMT puncta and Hsp60 quantified in at least 50 cells per condition per independent experiment. The co-localization percentage was calculated using the double positive foci/MGMT + foci. * P < 0.05. ( D ) BMMs from WT mice were stimulated with poly (I:C) as described above, and cytoplasmic and nuclear fractionation was performed. Lysates were subjected to Western blotting to detect MGMT, β-tubulin and histone proteins.

Article Snippet: The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 μg/mL in blocking solution at 4 °C overnight.

Techniques: Incubation, Staining, Marker, Confocal Microscopy, Fractionation, Western Blot

Journal: Scientific Reports

Article Title: Loss of O 6 -methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

doi: 10.1038/s41598-024-78885-3

Figure Lengend Snippet: Loss of MGMT increases ROS and mtROS production in BMMs. ( A ) WT and MGMT-KO BMMs were treated with bafilomycin A1 (500 nM), poly (I:C) (20 µg/ml) or poly (I:C) with bafilomycin A1 and incubated for 24 h. After incubation, the cells were treated with DCFH-DA (10 µM) for 15 min, washed with PBS, and the fluorescence intensity of the ROS was immediately measured at 498/520 nM via a microplate reader. ( B , C ) Cells were fixed and stained for HSP60 and nuclei and were subsequently stained with MitoSOX reagent (5 µM) for 10 min. The cells were processed for confocal microscopy analysis. Scale bar = 2 µM. ( C ) Corrected total cell fluorescence (CTCF) of MitoSOX was performed on the basis of the images obtained from ( B ). The data are presented as the means ± SEMs from three independent experiments. *** P < 0.001, ** P < 0.01 and * P < 0.05.

Article Snippet: The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 μg/mL in blocking solution at 4 °C overnight.

Techniques: Incubation, Fluorescence, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: Loss of O 6 -methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

doi: 10.1038/s41598-024-78885-3

Figure Lengend Snippet: Loss of MGMT enhances mitophagy in macrophages. ( A , B ) WT and MGMT KO BMMs were treated with bafilomycin A1 (500 nM) and incubated for 24 h. The cells were fixed and stained for HSP60 and LC3B and processed for confocal microscopy analysis. The colocalization of HSP60 and LC3B was quantified in at least 50 cells per condition per independent experiment. The data are presented as the means ± SEMs from three independent experiments. Scale bar = 5 µM. *** P < 0.001, ** P < 0.01 and * P < 0.05. ( C , D ) Cells were treated as described above and incubated with LysoTracker Deep Red (100 nM) for 1 h. The cells were then fixed and stained for HSP60 and processed for confocal microscopy analysis. At least 50 cells per condition per independent experiment were quantified for colocalization between lysosomes and HSP60. The data are presented as the means ± SEMs from three independent experiments. *** P < 0.001, ** P < 0.01 and * P < 0.05.

Article Snippet: The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 μg/mL in blocking solution at 4 °C overnight.

Techniques: Incubation, Staining, Confocal Microscopy