rabbit anti timp1  (Bioss)

Journal: Acta Pharmaceutica Sinica. B

Article Title: NAT10 inhibition alleviates astrocyte autophagy by impeding ac4C acetylation of Timp1 mRNA in ischemic stroke

doi: 10.1016/j.apsb.2025.03.042

Figure Lengend Snippet: NAT10 enhances Timp1 mRNA translation via ac4C modification. (A) Experimental procedure and timeline for RNC-seq experiment. Peri-infarct cortex for RNC-seq was pooled from 8 animals and defined as one sample. There were 3 samples for each group. (B) Venn diagram showing the number of genes with significant changes in expression (up: fold change ≥2, P < 0.05; down: fold change ≤0.5, P < 0.05). (C) Relative expression of Timp1 in the peri-infarct cortex as determined by qPCR. NAT10 was inhibited by remodelin administration on Days 3–7 after stroke and the level of Timp1 was examined on Day 8 after the PT stroke. n = 6 animals/group. ∗∗∗ P < 0.001 vs. Sham + Vehicle group, one-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (D) Representative immunoblots of TIMP1 expression in peri-infarct cortex. NAT10 was inhibited by remodelin administration on Days 3–7 after stroke and the expression of TIMP1 was examined by Western blot on Day 8 after stroke. n = 6 animals/group. ∗∗∗ P < 0.001 vs. Sham + Vehicle group; ### P < 0.001 versus PT + Vehicle group; one-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (E) Polysome assay showing the translation state of Timp1 mRNA in primary mouse astrocytes. The vehicle or remodelin pre-treated astrocytes were subjected to OGD for 6 h. n = 3. ∗∗∗ P < 0.001 vs. the Con + Vehicle group; ### P < 0.001 vs. the OGD + Vehicle group; two-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (F) The predicted ac4C motif (CXXCXXCXX, where X represents A, G, C, or U) location in the CDS of Timp1 mRNA. The forward and reverse arrows represented paired qPCR primers. a–c indicated the 3 ac4C motif regions in the Timp1 CDS. F and R indicate the designed PCR primer pairs for amplifying the specific ac4C motifs. (G) Detection of the 3 corresponding ac4C motifs via RNA immunoprecipitation (RIP)-PCR using the 3 PCR primer pairs. M indicated marker. (H) The effect of NAT10 upregulation on the ac4C level of Timp1 CDS (c motif sites). Cultured primary mouse astrocytes were transduced with lentivirus expressing Nat10 or the Gfp control for 48 h and the enrichment of ac4C on Timp1 CDS was examined via RIP-qPCR with anti-ac4C. n = 3. ∗∗ P < 0.01 vs. the Gfp group, Student's t -test. (I) The effect of NAT10 downregulation on the ac4C level of Timp1 CDS (c motif sites). Cultured primary mouse astrocytes were transduced with lentivirus expressing shNat10 or shScr control for 48 h and the enrichment of ac4C on Timp1 CDS was examined via RIP-qPCR with anti-ac4C. n = 3. ∗ P < 0.05 vs. the shScr group, Student's t -test. All data are presented as the mean ± SEM.

Article Snippet: After separation on sodium dodecyl sulfate-polyacrylamide gels (10% and 12%), the proteins were transferred onto polyvinylidene fluoride membranes with electrophoretic equipment and incubated overnight at 4 °C with rabbit anti-NAT10 antibody (1:1000), mouse anti-NeuN antibody (1:1000), mouse anti-GFAP antibody (1:1000), rabbit anti-C3 antibody (1:1000, ab200999, Abcam), rabbit anti-p62 antibody (1:1000, 18420-1-AP, Proteintech), rabbit anti-LC3B antibody (1:1000, L7543, Sigma–Aldrich), rabbit anti-TIMP1 antibody (1:1000, 26847-1-AP, Proteintech), rabbit anti- β -tubulin antibody (1:3000, 66240-1-Ig, Proteintech) or mouse anti- β -actin antibody (1:3000, 66009-1-lg, Proteintech).

Techniques: Modification, Expressing, Western Blot, RNA Immunoprecipitation, Marker, Cell Culture, Transduction, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: NAT10 inhibition alleviates astrocyte autophagy by impeding ac4C acetylation of Timp1 mRNA in ischemic stroke

doi: 10.1016/j.apsb.2025.03.042

Figure Lengend Snippet: NAT10 regulates astrocyte autophagy via TIMP1. (A) Representative immunoblots of TIMP1 expression in cultured astrocytes with/without Timp1 siRNA transfection. Cultured primary mouse astrocytes were transfected with siRNA for 24 h. n = 4. ∗∗ P < 0.01 versus the Scr group, Student's t -test. (B) The effect of Timp1 siRNA on the expression of LC3B-II in OGD-treated cells. n = 3. ∗∗∗ P < 0.001 vs. the Con + Scr group; # P < 0.05 vs. the OGD + Scr group; two-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (C) The effect of Nat10 and Timp1 siRNA on the expression of LC3B-II under OGD treatment. Lentiviruses expressing Nat10 and Timp1 siRNA were co-transduced into primary mouse astrocytes. After 48 h, the cells were subjected to OGD for 6 h. n = 3. ∗∗ P < 0.01 vs. the Gfp + Scr group; # P < 0.05 vs. the Nat10+Scr group; two-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (D) Cartoon showing CRISPR-dCasRx “writing” ac4C to the given CDS in Timp1 mRNA. gRNA, small guide RNA. 511 to 530 (gRNA-511) or 612 to 631 (gRNA-612) represented the location of the c ac4C motif sites in the Timp1 CDS. (E, F) Analysis of ac4C levels in the Timp1 CDS 48 h after co-transduction of CRISPR-dCasRx-Nat10 and gRNA-511 or gRNA-612 into primary mouse astrocytes. n = 3. ∗∗∗ P < 0.001 vs. the Vector group, one-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (G) The effect of CRISPR-dCasRx-Nat10 and gRNA-511 or gRNA-612 on the expression of TIMP1 and LC3B-II. CRISPR-dCasRx-Nat10 and gRNA-511 or gRNA-612 were co-transduced into primary mouse astrocytes for 48 h before collection for analysis. n = 3. ∗ P < 0.05 vs. the Vector group, one-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. All data are presented as the mean ± SEM.

Article Snippet: After separation on sodium dodecyl sulfate-polyacrylamide gels (10% and 12%), the proteins were transferred onto polyvinylidene fluoride membranes with electrophoretic equipment and incubated overnight at 4 °C with rabbit anti-NAT10 antibody (1:1000), mouse anti-NeuN antibody (1:1000), mouse anti-GFAP antibody (1:1000), rabbit anti-C3 antibody (1:1000, ab200999, Abcam), rabbit anti-p62 antibody (1:1000, 18420-1-AP, Proteintech), rabbit anti-LC3B antibody (1:1000, L7543, Sigma–Aldrich), rabbit anti-TIMP1 antibody (1:1000, 26847-1-AP, Proteintech), rabbit anti- β -tubulin antibody (1:3000, 66240-1-Ig, Proteintech) or mouse anti- β -actin antibody (1:3000, 66009-1-lg, Proteintech).

Techniques: Western Blot, Expressing, Cell Culture, Transfection, CRISPR, Transduction, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: NAT10 inhibition alleviates astrocyte autophagy by impeding ac4C acetylation of Timp1 mRNA in ischemic stroke

doi: 10.1016/j.apsb.2025.03.042

Figure Lengend Snippet: TIMP1 mediates the detrimental effect of NAT10 on functional recovery in PT mice. (A) Schematic of the experimental timeline. Lentivirus (LV; LV-CRISPR-dCasRx-Nat10 plus LV-gRNA-511 or LV-gRNA-612) was injected into the prospective stroke site in the cortex 7 days prior to stroke. Behavioral performances were examined at the pre-stroke baseline and on Day 7 after stroke. Nissl staining was performed on Day 8 after stroke. (B, C) Representative Nissl-stained sections from PT + scrambled + CRISPR-dCasRx-Nat10, PT + gRNA-511+CRISPR-dCasRx-Nat10, or PT + gRNA-612+CRISPR-dCasRx-Nat10 mice on Day 8 post-stroke. n = 7 animals/group. ∗∗ P < 0.01 vs. the PT + Scr + CRISPR-dCasRx-Nat10 group, one-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (D–F) The effect of co-microinjection of CRISPR-dCasRx-Nat10 and gRNA-511 or gRNA-612 on behavioral recovery on Day 7 after stroke, as measured by the grid-walking test (D), cylinder test of forelimb function (E), and adhesive removal test (F). n = 7 or 8 animals/group. ∗∗∗ P < 0.001 vs. the Sham + PBS group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the PT + Scr + dCasRx-Nat10 group; two-way repeated-measures ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. (G, H) Immunoreactivity for astrocytic marker GFAP under different treatment conditions on Day 8 after stroke. n = 6 animals/group. ∗ P < 0.05, ∗∗ P < 0.01 vs. PT + Scr + CRISPR-dCasRx-Nat10 group, one-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. Scale bar, 100 μm. (I, J) The effect of co-microinjection of CRISPR-dCasRx-Nat10 and gRNA-511 or gRNA-612 on the expression of GFAP, LC3B-II and p62. The tissue in the peri-infarct region was collected on Day 8 after injury. n = 6 animals/group. ∗ P < 0.05, ∗∗∗ P < 0.001 vs. the Sham + PBS group; # P < 0.05, ## P < 0.01 vs. the PT + Scr + dCasRx-Nat10 group; two-way ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. All data are presented as the mean ± SEM.

Article Snippet: After separation on sodium dodecyl sulfate-polyacrylamide gels (10% and 12%), the proteins were transferred onto polyvinylidene fluoride membranes with electrophoretic equipment and incubated overnight at 4 °C with rabbit anti-NAT10 antibody (1:1000), mouse anti-NeuN antibody (1:1000), mouse anti-GFAP antibody (1:1000), rabbit anti-C3 antibody (1:1000, ab200999, Abcam), rabbit anti-p62 antibody (1:1000, 18420-1-AP, Proteintech), rabbit anti-LC3B antibody (1:1000, L7543, Sigma–Aldrich), rabbit anti-TIMP1 antibody (1:1000, 26847-1-AP, Proteintech), rabbit anti- β -tubulin antibody (1:3000, 66240-1-Ig, Proteintech) or mouse anti- β -actin antibody (1:3000, 66009-1-lg, Proteintech).

Techniques: Functional Assay, CRISPR, Injection, Staining, Microinjection, Adhesive, Marker, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: NAT10 inhibition alleviates astrocyte autophagy by impeding ac4C acetylation of Timp1 mRNA in ischemic stroke

doi: 10.1016/j.apsb.2025.03.042

Figure Lengend Snippet: Downregulation of astrocytic TIMP1 promotes functional recovery in PT mice. (A) Experimental procedure and timeline. The astrocyte-targeting AAV-shRNA-Timp1 or AAV-shRNA-Scr was microinjected into the prospective stroke site in the cortex on Day 28 before stroke. Behavioral performance was examined at the pre-stroke baseline and on Day 7 after stroke followed by Nissl staining. (B) The level of TIMP1 was measured by Western blot. The cortex was collected on Day 28 after microinjection. n = 3 animals/group. ∗∗∗ P < 0.001 vs. the shRNA-Scr group, Student's t -test. (C) Representative images of AAV-shRNA-Timp1 (red) infection of astrocytes (green). Scale bar, 50 μm. (D, E) Representative Nissl-stained sections from PT + shRNA-Scr or PT + shRNA-Timp1 mice on Day 8 post-stroke. n = 6 animals/group. ∗∗∗ P < 0.001 vs. the PT + shRNA-Scr group, Student's t -test. (F–H) TIMP1 inhibition in astrocytes improved behavioral recovery on Day 7 after stroke as measured by the grid-walking test (F), the cylinder test of forelimb function (L indicated the left forepaw, R indicated the right forepaw) (G), and the adhesive removal test (H). n = 9 animals/group. ∗∗∗ P < 0.001 vs. the sham + shRNA-Scr group; # P < 0.05, ### P < 0.001 vs. the PT + shRNA-Scr group; two-way repeated-measures ANOVA followed by Holm–Sidak post hoc multiple-comparisons test. All data are presented as the mean ± SEM.

Article Snippet: After separation on sodium dodecyl sulfate-polyacrylamide gels (10% and 12%), the proteins were transferred onto polyvinylidene fluoride membranes with electrophoretic equipment and incubated overnight at 4 °C with rabbit anti-NAT10 antibody (1:1000), mouse anti-NeuN antibody (1:1000), mouse anti-GFAP antibody (1:1000), rabbit anti-C3 antibody (1:1000, ab200999, Abcam), rabbit anti-p62 antibody (1:1000, 18420-1-AP, Proteintech), rabbit anti-LC3B antibody (1:1000, L7543, Sigma–Aldrich), rabbit anti-TIMP1 antibody (1:1000, 26847-1-AP, Proteintech), rabbit anti- β -tubulin antibody (1:3000, 66240-1-Ig, Proteintech) or mouse anti- β -actin antibody (1:3000, 66009-1-lg, Proteintech).

Techniques: Functional Assay, shRNA, Staining, Western Blot, Microinjection, Infection, Inhibition, Adhesive

Journal: Molecular Medicine

Article Title: TIMP1 regulates ferroptosis in osteoblasts by inhibiting TFRC ubiquitination: an in vitro and in vivo study

doi: 10.1186/s10020-024-01000-9

Figure Lengend Snippet: TIMP1 is involved in the regulation of ferroptosis in osteoblasts in vitro. ( A ) Bioinformatics analysis of differentially expressed genes in RNA-sequencing of control and HGHF-treated osteoblasts. ( B ) qRT-PCR and WB analysis of TIMP1 mRNA and protein expression levels in LV-shTIMP1 and LV-NC groups. ( C ) WB analysis of PTGS2, GPX4, and TFRC protein expression levels, with WB result analysis (normalized to β-Actin control band intensity, n = 3). ( D ) Quantitative measurement of MDA levels in osteoblasts using an MDA assay kit. ( E ) BODIPY 581/591 C11 probe estimation of lipid peroxidation levels in osteoblasts (assessed by flow cytometry). ( F ) FerroOrange staining to estimate intracellular Fe 2+ levels (assessed by flow cytometry). ( G ) Flow cytometric estimation of ROS levels in osteoblasts. ( H ) Two-photon laser confocal microscopy images showing Fe 2+ levels in osteoblasts after FerroOrange staining. Data are presented as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001, ns, no statistical significance)

Article Snippet: The next day, the immunoprecipitated proteins were washed three times with PBS and boiled in 1× loading buffer for 5 min. Western blotting was performed using antibodies against Ubiquitin (Cell Signaling Technology, 20326), TIMP1 (Cell Signaling Technology, 63363), and TFRC (Abcam, AB214039).

Techniques: In Vitro, RNA Sequencing, Control, Quantitative RT-PCR, Expressing, Multiple Displacement Amplification, Flow Cytometry, Staining, Confocal Microscopy

Journal: Molecular Medicine

Article Title: TIMP1 regulates ferroptosis in osteoblasts by inhibiting TFRC ubiquitination: an in vitro and in vivo study

doi: 10.1186/s10020-024-01000-9

Figure Lengend Snippet: TIMP1 Regulates Osteoblast Ferroptosis via TFRC. ( A ) qRT-PCR and WB analysis of TFRC mRNA and protein expression levels in oe-TFRC and oe-NC groups. ( B ) Quantitative measurement of MDA levels in osteoblasts using an MDA assay kit. ( C ) WB analysis of PTGS2, GPX4, TFRC, and TIMP1 protein expression levels. ( D ) WB result analysis (normalized to β-Actin control band intensity, n = 3). ( E ) FerroOrange staining to estimate intracellular Fe 2+ levels (assessed by flow cytometry). ( F ) Flow cytometric estimation of ROS levels in osteoblasts. ( G ) BODIPY 581/591 C11 probe estimation of lipid peroxidation levels in osteoblasts (assessed by flow cytometry). ( H ) Two-photon laser confocal microscopy images showing Fe 2+ levels in osteoblasts after FerroOrange staining. Data are presented as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001, ns, no statistical significance)

Article Snippet: The next day, the immunoprecipitated proteins were washed three times with PBS and boiled in 1× loading buffer for 5 min. Western blotting was performed using antibodies against Ubiquitin (Cell Signaling Technology, 20326), TIMP1 (Cell Signaling Technology, 63363), and TFRC (Abcam, AB214039).

Techniques: Quantitative RT-PCR, Expressing, Multiple Displacement Amplification, Control, Staining, Flow Cytometry, Confocal Microscopy

Journal: Molecular Medicine

Article Title: TIMP1 regulates ferroptosis in osteoblasts by inhibiting TFRC ubiquitination: an in vitro and in vivo study

doi: 10.1186/s10020-024-01000-9

Figure Lengend Snippet: TIMP1 influences ferroptosis in osteoblasts by regulating the stability of TFRC protein. ( A ) qRT-PCR analysis of TIMP1 mRNA expression levels in LV-shTIMP1 and LV-NC groups. ( B ) Co-IP results showing immunoprecipitation of total cell lysates with protein A/G agarose beads (mock IgG) or anti-TFRC antibody in control and HGHF-treated osteoblasts, followed by immunoblotting with the indicated antibodies. ( C ) Schematic representation of TIMP1-TFRC molecular docking. ( D ) Two-photon laser confocal microscopy images showing immunofluorescence of TIMP1 (red) and TFRC (green) in control and HGHF-treated osteoblasts. ( E ) IP analysis of cell lysates using anti-TIMP1 antibody, followed by incubation with the indicated antibodies, including ubiquitination antibody, anti-TIMP1, and anti-TFRC, for WB analysis. ( F ) Schematic representation of the mechanism by which TIMP1-TFRC regulates osteoblast ferroptosis, leading to osteoporosis

Article Snippet: The next day, the immunoprecipitated proteins were washed three times with PBS and boiled in 1× loading buffer for 5 min. Western blotting was performed using antibodies against Ubiquitin (Cell Signaling Technology, 20326), TIMP1 (Cell Signaling Technology, 63363), and TFRC (Abcam, AB214039).

Techniques: Quantitative RT-PCR, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Confocal Microscopy, Immunofluorescence, Incubation, Ubiquitin Proteomics

Journal: Molecular Medicine

Article Title: TIMP1 regulates ferroptosis in osteoblasts by inhibiting TFRC ubiquitination: an in vitro and in vivo study

doi: 10.1186/s10020-024-01000-9

Figure Lengend Snippet: TIMP1 regulates osteoblast ferroptosis and diabetic osteoporosis. ( A ) Schematic representation of the grouping and lentiviral intervention in the type 2 diabetic mouse model. ( B ) Histological images of distal femoral tissue sections stained with H&E. ( C ) WB analysis of GPX4 expression levels in femoral bone tissue ( n = 4), with semi-quantitative analysis based on WB results. ( D ) 2D and 3D micro-CT images of the distal femur in LV-NC, STZ&HFD + LV-NC, and STZ&HFD + LV-shTIMP1 groups of mice. ( E ) Quantitative analysis of trabecular bone density (BMD) at the distal femur in mice ( n = 4). ( F ) Quantitative analysis of BV/TV, Tb.Th, Tb.Sp, and Tb.N in the trabecular bone of the distal femur in mice ( n = 4). ( G ) Immunohistochemical images showing ACSL4, GPX4, PTGS2, TFRC, and TIMP1 expression in control and STZ&HFD mice ( n = 4). Data are presented as mean ± SEM

Article Snippet: The next day, the immunoprecipitated proteins were washed three times with PBS and boiled in 1× loading buffer for 5 min. Western blotting was performed using antibodies against Ubiquitin (Cell Signaling Technology, 20326), TIMP1 (Cell Signaling Technology, 63363), and TFRC (Abcam, AB214039).

Techniques: Staining, Expressing, Micro-CT, Immunohistochemical staining, Control