Journal: Biochemistry and Biophysics Reports

Article Title: Normal spermatogenesis and fertility in Spmip8 deficiency male mice

doi: 10.1016/j.bbrep.2025.102406

Figure Lengend Snippet: Generation and validation of Spmip8 knockout mice. (A) Schematic diagram of Spmip8 −/− mouse creation. (B) Sanger sequencing of genomic DNA shows a deletion in the Spmip8 - gene. (C) Spmip8 −/− mice were identified by genomic PCR. (D) Spmip8 - transcripts were not detected in adult Spmip8 −/− testes, n = 3 for each genotype. (E) Western blot analysis the SPMIP8 protein in Spmip8 knockout mice. α-TUBULIN was used as a loading control. (F) Immunofluorescence staining of SPMIP8 (green), PNA (acrosome, red) in testis sections from 10-week-old WT and Spmip8 −/− mice. Magnification ×40 in the panels. DAPI (blue) stains the nuclei. The head signal in elongating spermatids is non-specific, as it appears in both WT and Spmip8 −/− testis sections. Scale bar: 50 μm ∗∗∗ P < 0.001.

Article Snippet: The membrane was blocked with 5 % nonfat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies against SPMIP8 (1:1000, HPA062092, Sigma, Germany) and α-TUBULIN (1:5000, 11224-1-AP, Proteintech, China).

Techniques: Biomarker Discovery, Knock-Out, Sequencing, Western Blot, Control, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: Genetic suppression of GCN2 reverses developmental delay and seizure phenotypes in dPARS2-deficient flies. (A) Western blot analysis of P-GCN2, GCN2 and P-PERK in protein extracts from control and elav- Gal4-driven dPARS2 knockdown fly heads. α-tubulin was used as a loading control. (B) Quantification of the Western blots shown in A. P-GCN2, N = 3; GCN2 and P-PERK, N = 4. ∗∗p < 0.01, ns, not significant. (C) Western blot analysis of P-eIF2α in protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown fly heads. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 3. ∗p < 0.05, ∗∗p < 0.01. (E) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown fly heads. Flies were fed with puromycin. α-actin was used as the loading control. (F) Quantification of the Western blots shown in E. N = 3, ∗p < 0.05, ∗∗∗p < 0.001. (G) Images of control, elav -Gal4-driven dPARS2 knockdown and elav -Gal4-driven dPARS2 and GCN2 double knockdown flies at different developmental stages. Scale bars: 500 μm. (H) Graph showing pupariation rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown larvae. N = 3, n = 28–30. (I) Graph showing eclosion rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown pupae. N = 3, n = 28–30. (J) Graph showing percentage of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies displaying Bang-sensitive paralytic phenotypes. N = 3, n = 10 sample. ∗∗∗∗p < 0.0001. (K) Graph showing the recovery time of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies from paralysis. n = 30. ∗∗∗∗p < 0.0001.

Article Snippet: Primary antibodies used were anti-MT-ND1 (Abcam, AB181848-1001), anti-MT- CO2 (Proteintech, 55070-1-AP), anti-MT-ATP8 (Proteintech, 26723-1-AP),anti-NDUFS1 (Proteintech, 12444-1-AP), anti-NDUFS3 (Abcam, ab14711), anti-UQCRFS1 (Abcam, ab14746), anti-ATP5A (Abcam, ab14748), anti-SDHB (Proteintech, 10620-1-AP), anti-Porin/VDAC (Abcam, ab14734), anti-P-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 2103), anti-P-PERK (ABclonal, AP0886), anti-PERK (ABclonal, A27664 ), anti-P-GCN2 (Abcam, ab75836), anti-GCN2 (ABclonal, A2307), anti-LDH (ThermoFisher, PA5-26531), anti-PARS2 (ABclonal, A16512), anti-His (yeasen, 30405ES50), anti-ATF4 (Abcam, ab1371), anti-Alpha actin (Proteintech, 23660-1-AP) and anti-Alpha tubulin (Proteintech, 66031-1-Ig).

Techniques: Western Blot, Control, Knockdown, Staining

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: PARS2 V95I mutation causes mitochondrial dysfunction and ISR activation in human cells (A) Western blot analysis of ectopically expressed PARS2 proteins. Lysates from HEK-293T cells transfected with plasmids encoding His-tagged wild-type (WT) or the indicated PARS2 variants were immunoblotted with an anti-His antibody. α-actin was used as a loading control. (B) Quantification of the Western blots shown in A. N = 5, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Western blot analysis of endogenous PARS2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 4, ∗∗∗p < 0.001. (E) Western blot analysis of mtDNA-encoded CO2 and ATP8 and nuclear-DNA encoded NDUFS1, NDUFS3, UQCRFS1 and ATP5A in protein extracts from the wild-type controls and the PARS2 V95I cells. VDAC was used as a loading control. (F) Quantification of the Western blots shown in E. MT-CO2, MT-ATP8, NDUFS1, NDUFS3, and ATP5A, N = 4; UQCRFS1, N = 7. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant. (G) CI, CII and CIV in-gel activity analysis of isolated mitochondria from the wild-type controls and the PARS2 V95I cells. (H) Western blot analysis of P-eIF2α and eIF2α in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (I) Quantification of the Western blots shown in H. N = 5, ∗∗∗∗p < 0.0001. (J) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as the loading control. (K) Quantification of the Western blots shown in J. N = 4. ∗∗∗∗p < 0.0001. (L) Western blot analysis of ATF4 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (M) Quantification of the Western blots shown in L. N = 5, ∗∗∗p < 0.001. (N) Western blot analysis of P-GCN2 and GCN2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (O) Quantification of the Western blots shown in N. N = 4, ∗∗p < 0.01. (P) Western blot analysis of P-PERK and PERK in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (Q) Quantification of the Western blots shown in P. N = 5, ns, not significant.

Article Snippet: Primary antibodies used were anti-MT-ND1 (Abcam, AB181848-1001), anti-MT- CO2 (Proteintech, 55070-1-AP), anti-MT-ATP8 (Proteintech, 26723-1-AP),anti-NDUFS1 (Proteintech, 12444-1-AP), anti-NDUFS3 (Abcam, ab14711), anti-UQCRFS1 (Abcam, ab14746), anti-ATP5A (Abcam, ab14748), anti-SDHB (Proteintech, 10620-1-AP), anti-Porin/VDAC (Abcam, ab14734), anti-P-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 2103), anti-P-PERK (ABclonal, AP0886), anti-PERK (ABclonal, A27664 ), anti-P-GCN2 (Abcam, ab75836), anti-GCN2 (ABclonal, A2307), anti-LDH (ThermoFisher, PA5-26531), anti-PARS2 (ABclonal, A16512), anti-His (yeasen, 30405ES50), anti-ATF4 (Abcam, ab1371), anti-Alpha actin (Proteintech, 23660-1-AP) and anti-Alpha tubulin (Proteintech, 66031-1-Ig).

Techniques: Mutagenesis, Activation Assay, Western Blot, Transfection, Control, Activity Assay, Isolation, Staining

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: Rabeprazole modulates SMAD3 phosphorylation and nuclear translocation. (A) GES-1 and AGS cells were treated with or without rabeprazole for 1 h, and the phosphorylation of SMAD3 linker was detected by immunoblotting. (B-E) The band intensities were quantified and analyzed by one sample t-test. Data are shown as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001, n=3. (F) Left panel: The subcellular fraction was isolated using nuclear and cytoplasmic protein extraction kit according to manufacturer's instructions. The SMAD3 level was detected by western blotting, α-tubulin and lamin A/C were used as cytosolic and nuclear internal controls. Right panel: IF analysis of SMAD3 in AGS cells treated with or without rabeprazole for 1 h. Scale bar, 100 µm. SMAD3, SMAD family member 3; IF, immunofluorescence; phospho, phosphorylated.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Isolation, Protein Extraction, Immunofluorescence

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: Genetic suppression of GCN2 reverses developmental delay and seizure phenotypes in dPARS2-deficient flies. (A) Western blot analysis of P-GCN2, GCN2 and P-PERK in protein extracts from control and elav- Gal4-driven dPARS2 knockdown fly heads. α-tubulin was used as a loading control. (B) Quantification of the Western blots shown in A. P-GCN2, N = 3; GCN2 and P-PERK, N = 4. ∗∗p < 0.01, ns, not significant. (C) Western blot analysis of P-eIF2α in protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown fly heads. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 3. ∗p < 0.05, ∗∗p < 0.01. (E) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown fly heads. Flies were fed with puromycin. α-actin was used as the loading control. (F) Quantification of the Western blots shown in E. N = 3, ∗p < 0.05, ∗∗∗p < 0.001. (G) Images of control, elav -Gal4-driven dPARS2 knockdown and elav -Gal4-driven dPARS2 and GCN2 double knockdown flies at different developmental stages. Scale bars: 500 μm. (H) Graph showing pupariation rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown larvae. N = 3, n = 28–30. (I) Graph showing eclosion rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown pupae. N = 3, n = 28–30. (J) Graph showing percentage of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies displaying Bang-sensitive paralytic phenotypes. N = 3, n = 10 sample. ∗∗∗∗p < 0.0001. (K) Graph showing the recovery time of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies from paralysis. n = 30. ∗∗∗∗p < 0.0001.

Article Snippet: Mouse anti-Alpha tubulin , Proteintech , 66031-1-Ig.

Techniques: Western Blot, Control, Knockdown, Staining

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: PARS2 V95I mutation causes mitochondrial dysfunction and ISR activation in human cells (A) Western blot analysis of ectopically expressed PARS2 proteins. Lysates from HEK-293T cells transfected with plasmids encoding His-tagged wild-type (WT) or the indicated PARS2 variants were immunoblotted with an anti-His antibody. α-actin was used as a loading control. (B) Quantification of the Western blots shown in A. N = 5, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Western blot analysis of endogenous PARS2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 4, ∗∗∗p < 0.001. (E) Western blot analysis of mtDNA-encoded CO2 and ATP8 and nuclear-DNA encoded NDUFS1, NDUFS3, UQCRFS1 and ATP5A in protein extracts from the wild-type controls and the PARS2 V95I cells. VDAC was used as a loading control. (F) Quantification of the Western blots shown in E. MT-CO2, MT-ATP8, NDUFS1, NDUFS3, and ATP5A, N = 4; UQCRFS1, N = 7. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant. (G) CI, CII and CIV in-gel activity analysis of isolated mitochondria from the wild-type controls and the PARS2 V95I cells. (H) Western blot analysis of P-eIF2α and eIF2α in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (I) Quantification of the Western blots shown in H. N = 5, ∗∗∗∗p < 0.0001. (J) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as the loading control. (K) Quantification of the Western blots shown in J. N = 4. ∗∗∗∗p < 0.0001. (L) Western blot analysis of ATF4 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (M) Quantification of the Western blots shown in L. N = 5, ∗∗∗p < 0.001. (N) Western blot analysis of P-GCN2 and GCN2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (O) Quantification of the Western blots shown in N. N = 4, ∗∗p < 0.01. (P) Western blot analysis of P-PERK and PERK in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (Q) Quantification of the Western blots shown in P. N = 5, ns, not significant.

Article Snippet: Mouse anti-Alpha tubulin , Proteintech , 66031-1-Ig.

Techniques: Mutagenesis, Activation Assay, Western Blot, Transfection, Control, Activity Assay, Isolation, Staining