pcdna3 myc  (Addgene inc)

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: Bach1 inhibition alleviates SNI-induced pain and ferroptosis in the spinal cord. (A) Western blot analysis and quantification of Bach1 protein expression in the spinal cord at 3, 7, and 14 days after SNI (n = 6 per group). (B) Double immunofluorescence staining of Bach1 (red) with the cellular markers NeuN (neurons, green), GFAP (astrocytes, green), and Iba1 (microglia, green) in the spinal dorsal horn of SNI mice (n = 3 per group). Scale bar: 100 μm. White boxes indicate representative cells shown at higher magnification. (C) Behavioral assessments (PWT and PWL) in SNI mice treated with the Bach1 inhibitor Bach1-IN-1 or vehicle (n = 6 per group). (D-G) Representative blots and quantitative analysis of Bach1, GPX4 and SLC7A11 protein expression in the spinal cord following Bach1-IN-1 treatment (n = 6 per group). (H–I) Biochemical assays of Fe 2+ and MDA levels in spinal cord tissues following Bach1-IN-1 treatment (n = 6 per group). (J) Behavioral assessment following intrathecal injection of siBach1 or siNC control (n = 9 per group). (K) Western blot analysis of 4-HNE following siBach1 treatment (n = 6 per group). (L) Representative DHE staining (red) in the spinal dorsal horn to assess superoxide production (n = 3 per group). Scale bar: 100 μm. (M-Q) Western blot analysis of Bach1, ACSL4, SLC7A11, and GPX4 following siBach1 treatment (n = 6 per group). (R-T) Biochemical analysis of Fe 2+ , MDA, and GSH levels (n = 6 per group). (U) Representative TEM images and quantification of spinal cord mitochondria following siBach1 treatment (n = 3 per group). Scale bar: 500 nm. Data are presented as mean ± SEM. Significance was determined by one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham group or Sham + Vehicle group or Sham + siNC group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + Vehicle group or SNI + siNC group). The value of n represents the number of independent biological samples. All molecular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

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

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: NOX4 mediates ferroptosis and pain hypersensitivity in SNI mice. (A-B) Western blot analysis of NOX4 expression in the spinal cord following treatment with Bach1-IN-1 or siBach1 (n = 6 per group). (C) Behavioral assessment of PWT and PWL in mice treated with siNOX4 and the ferroptosis inducer RSL3 (n = 9 per group). (D-I) Representative Western blot images and quantification of NOX4, GPX4, SLC7A11, ACSL4, and 4-HNE in the spinal cord of mice receiving the indicated treatments (n = 6 per group). (J-L) Biochemical analysis of Fe 2+ , MDA, and GSH levels in spinal cord (n = 6 per group). (M) TEM analysis of mitochondrial ultrastructure in spinal cord neurons. Scale bar: 500 nm (n = 3 per group). Data analysis used one-way or two-way ANOVA followed by Bonferroni's post-hoc tests. Data are mean ± SEM (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham + Vehicle group or Sham + siNC group or Sham + siNC + Vehicle group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + Vehicle group or SNI + siNC group or SNI + siNC + Vehicle group; & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001 vs. SNI + siNOX4 + Vehicle group). The value of n represents the number of independent biological samples. All molecular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Western Blot, Expressing

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: Bach1 transcriptionally activates NOX4 to promote ferroptosis in the spinal cord. (A) Schematic of the potential Bach1-binding site on the NOX4 promoter, predicted using the JASPAR database. (B) Dual-luciferase reporter assay in N2a cells transfected with wild-type (WT) or mutant (MUT) NOX4 promoter constructs and Bach1 overexpression plasmids (∗∗∗∗ P < 0.0001, n = 3 per group). (C) ChIP-qPCR confirming the direct binding of Bach1 to the specific region of the NOX4 promoter (∗∗∗∗ P < 0.0001, n = 3 per group). (D) Representative fluorescence images showing EGFP expression in the spinal dorsal horn, confirming successful AAV transduction. Scale bar: 100 μm. (E) Behavioral assessments showing that AAV-mediated Bach1 overexpression reduced the PWT and PWL, and these effects were reversed by GLX351322 treatment (n = 6 per group). (F-J) Representative Western blots and quantitative analysis of Bach1, NOX4, SLC7A11, and GPX4 protein levels in the spinal cord. AAV-Bach1 increased NOX4 and decreased GPX4 and SLC7A11 expression, which were rescued by GLX351322 (n = 6 per group). (K–N) Biochemical assays showing that AAV-Bach1 elevated the levels of ROS, Fe 2+ , and MDA, and reduced GSH content in spinal cord tissues, and these changes were attenuated by GLX351322 (n = 6 per group). Data are presented as mean ± SEM. Comparisons between two independent groups were performed using unpaired two-tailed Student's t-tests. All multi-group comparisons were conducted using one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (E-N: ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Naïve + AAV-Vector group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. Naïve + AAV-Bach1 group). The value of n represents the number of independent biological samples. All molecular and cellular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Binding Assay, Luciferase, Reporter Assay, Transfection, Mutagenesis, Construct, Over Expression, ChIP-qPCR, Fluorescence, Expressing, Transduction, Western Blot, Two Tailed Test, Plasmid Preparation

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: Inhibition of USP7 alleviates Bach1 expression, ferroptosis and neuropathic pain. (A) Representative Western blots and quantitative analysis showing increased expression of USP7 in the spinal cord of SNI mice compared with sham controls (∗∗ P < 0.01, ∗∗∗ P < 0.001, n = 6 per group). (B) Double immunofluorescence staining of USP7 (red) with the cellular markers GFAP (astrocytes, green), Iba1 (microglia, green), and NeuN (neurons, green) in the spinal dorsal horn of SNI mice (n = 3 per group). White boxes indicate representative cells shown at higher magnification. Scale bar: 100 μm. (C) Behavioral assessments showing that intraperitoneal administration of the USP7 inhibitor P005091 significantly increased PWT and PWL in SNI mice compared with the SNI + Vehicle group (n = 6 per group). (D-I) Representative Western blots and quantitative analysis showing that P005091 treatment downregulated the expression of USP7, Bach1, and NOX4, and upregulated the expression of GPX4 and SLC7A11 in the spinal cord of SNI mice (n = 6 per group). (J-L) Biochemical assays showing that P005091 treatment reduced the SNI-induced elevations in Fe 2+ and MDA levels, and restored the content of GSH in spinal cord tissues (n = 6 per group). (M) Representative TEM images and quantification of spinal cord mitochondria. Scale bar: 500 nm. Data are presented as mean ± SEM. Significance was determined by one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham + Vehicle group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + Vehicle group). The value of n represents the number of independent biological samples. All molecular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Inhibition, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: USP7 stabilizes Bach1 and is required for its pro-ferroptotic activity in vivo. (A) Representative immunofluorescence images showing co-localization of USP7 (red) and Bach1 (green) in the spinal dorsal horn of mice. Scale bar: 100 μm. (B–C) Exogenous Co-IP analysis in N2a cells co-expressing FLAG-Bach1 and His-USP7 confirmed their specific interaction. (D) Endogenous Co-IP in spinal cord tissue using anti-USP7 antibody demonstrated that Bach1 co-precipitates with USP7 under physiological conditions. (E) Denaturing ubiquitination assay showing that USP7 overexpression markedly reduced HA-Ub-labeled Bach1 in N2a cells following MG132 treatment. (F) Quantification of CHX chase assay results demonstrates that USP7 overexpression extends the half-life of Bach1 from 3.43 h to 10.68 h, as shown by the representative immunoblot of Bach1 protein levels at 0, 2, 4, and 6 h (∗∗∗∗ P < 0.0001, n = 3 per group). (G) Representative EGFP fluorescence images confirming successful viral transduction in spinal dorsal horn. Scale bar: 100 μm. (H) Behavioral assessments of PWT and PWL (n = 9 per group). siUSP7 alleviated SNI-induced pain hypersensitivity, whereas Bach1 overexpression reversed this protective effect. (I-Q) Western blot analysis and quantification of USP7, Bach1, NOX4, GPX4, SLC7A11, ACSL4, and 4-HNE expression in spinal cord from indicated groups (n = 6 per group). (R–U) Biochemical measurements of Fe 2+ , MDA, GSH, and ATP levels in spinal cord tissues (n = 6 per group). (V) Representative TEM images and quantification showing mitochondrial morphology in spinal neurons (n = 3 per group). Scale bar: 500 nm. Data are presented as mean ± SEM. Comparisons between two independent groups were performed using unpaired two-tailed Student's t-tests. All multi-group comparisons were conducted using one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (H–V: ∗ P < 0.05, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham + AAV-Vector + siNC group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + AAV-Vector + siNC group; & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001 vs. SNI + AAV-Vector + siUSP7 group). The value of n represents the number of independent biological samples. All molecular and cellular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Activity Assay, In Vivo, Immunofluorescence, Co-Immunoprecipitation Assay, Expressing, Ubiquitin Proteomics, Over Expression, Labeling, Western Blot, Fluorescence, Transduction, Two Tailed Test, Plasmid Preparation

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: USP7 overexpression induces Bach1-dependent ferroptosis and pain hypersensitivity in naïve mice. (A) Representative fluorescence images showing EGFP expression in the spinal dorsal horn, confirming successful AAV-USP7 transduction. Scale bar: 100 μm. (B) Behavioral assessments showing that AAV-USP7 significantly decreased the PWT and PWL in naïve mice compared with the AAV-Vector control group (n = 6 per group). (C) RT-qPCR analysis of Bach1 mRNA levels in spinal tissues following USP7 overexpression (n = 6 per group). (D-F) Representative Western blots and quantitative analysis of spinal cord tissues showing increased protein levels of USP7 and Bach1 following AAV-USP7 injection (n = 6 per group). (G-H) Behavioral tests demonstrating that Bach1-IN-1 treatment reversed the reductions in PWT and PWL induced by AAV-USP7 overexpression (n = 6 per group). (I-L) Representative Western blots and quantitative analysis showing that AAV-USP7 increased NOX4 expression and decreased the levels of SLC7A11 and GPX4, which were restored by Bach1-IN-1 treatment (n = 6 per group). (M − O) Biochemical assays quantifying ferroptosis-related indicators in spinal cord tissues. AAV-USP7 elevated Fe 2+ and MDA levels and reduced GSH content, and these changes were attenuated by Bach1-IN-1 (n = 6 per group). Data are presented as mean ± SEM. Comparisons between two independent groups were performed using unpaired two-tailed Student's t-tests. All multi-group comparisons were conducted using one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Naïve + AAV-Vector group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. Naïve + AAV-USP7 group). The value of n represents the number of independent biological samples. All molecular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Over Expression, Fluorescence, Expressing, Transduction, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Injection, Two Tailed Test

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: USP7 promotes ferroptosis through Bach1 and forms a positive feedback loop with Bach1. (A-F) Representative Western blots and quantitative analysis of USP7, Bach1, NOX4, SLC7A11, and GPX4 protein levels in N2a cells following USP7 overexpression and/or Bach1 inhibition (n = 3 per group). (G) Flow cytometric analysis using the fluorescent probe BODIPY™ 581/591 C11 to detect lipid ROS. The ratio of oxidized (FITC) to total fluorescence was increased in OE-USP7 cells and reduced by co-treatment with Bach1-IN-1 (n = 3 per group). (H-J) Biochemical quantification of ferroptosis-related parameters in cell lysates. OE-USP7 elevated intracellular Fe 2+ and MDA levels and decreased GSH content; these changes were attenuated by Bach1-IN-1 (n = 3 per group). (K) Cell Counting Kit-8 (CCK-8) assay showing that Bach1-IN-1 restored the viability of OE-USP7 cells (n = 3 per group). (L) RT-qPCR analysis showing that USP7 mRNA levels were upregulated by Bach1 overexpression (OE-Bach1) and downregulated by Bach1 knockdown (si-Bach1) (n = 3 per group). (M − O) Representative Western blots and quantification of Bach1 and USP7 protein levels in N2a cells transfected with OE-Bach1 or si-Bach1, confirming the regulatory effect of Bach1 on USP7 expression (n = 3 per group). (P) JASPAR analysis of the USP7 promoter identified two putative Bach1-binding sites (MAREs) located at −1756 bp (Site 1) and −1159 bp (Site 2). (Q) Motif alignment revealed that Site 1 closely matches the consensus Bach1-binding sequence. (R) ChIP-qPCR analysis using primers flanking Site 1 (−1756 bp), confirming enrichment of Bach1 at this specific promoter locus in N2a cells (n = 3 per group). (S) Schematic of wild-type (WT) and mutant (MUT) USP7 promoter constructs used in luciferase reporter assays. (T) Luciferase assays show that Bach1 overexpression increases activity of the wild-type USP7 promoter, while mutation of the binding sites eliminates this effect (n = 3 per group). Data are presented as mean ± SEM. Comparisons between two independent groups were performed using unpaired two-tailed Student's t-tests. All multi-group comparisons were conducted using one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (B–K: ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 vs. Ctrl group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. OE-USP7 group; L-T:∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). The value of n represents the number of independent biological samples. All molecular and cellular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Western Blot, Over Expression, Inhibition, Fluorescence, Cell Counting, CCK-8 Assay, Quantitative RT-PCR, Knockdown, Transfection, Expressing, Binding Assay, Sequencing, ChIP-qPCR, Mutagenesis, Construct, Luciferase, Activity Assay, Two Tailed Test

Journal: ACS Omega

Article Title: Peptide Design for Targeting BTB Domain Homodimerization of BACH2: Complementary In Silico and In Vitro Approaches

doi: 10.1021/acsomega.6c01122

Figure Lengend Snippet: (A) Predicted relative binding free energies (RBFE) from MM-PBSA analysis of MD-simulated complexes and (B–D) amino acid-level contributions to RBFE for the positive control homodimer, BACH2 BTB domain monomer-control peptide (peptide 1), and BACH2 BTB domain monomer–peptide 13 complexes. Compared with the positive control (PC, BACH2 BTB homodimer, first bar), all BACH2 BTB monomer–peptide complexes exhibited higher RBFE values (numerically more negative values indicate an increase). Among these, the BACH2 BTB domain monomer–peptide 13 complex had the highest RBFE value (183.51 ± 9.61 kcal/mol (∼51 kcal/mol higher than PC on average) and the lowest p -value ( p < 0.0068). Other RBFE values were PC (BACH2 BTB homodimer): 234.90 ± 3.75 kcal/mol, BACH2–control peptide (peptide 1): 204.57 ± 9.98 kcal/mol (∼30 kcal/mol higher than PC), BACH2-peptide 4:207.57 ± 10.28 kcal/mol (∼27 kcal/mol higher than PC), BACH2–peptide 14:189.67 ± 12.23 kcal/mol (∼45 kcal/mol higher than PC), and BACH2–peptide 15:205.68 ± 8.93 kcal/mol (∼29 kcal/mol higher than PC). Although peptides 14 and 15 also had statistically significant p -values ( p < 0.05) compared with positive control, peptide 13 had the lowest p -value, making it the most suitable candidate for further in vitro studies. Additionally, peptide 13 showed the largest RBFE difference (∼50 kcal/mol higher than PC), indicating that it has the highest affinity toward the BTB domain. After identification of peptide 13 as the most plausible BACH BTB domain binder, its specificity toward the BACH BTB domain was tested using other BTB domain as negative controls. BACH1 BTB domain monomer-peptide 13 and BCL-6 BTB domain monomer–peptide 13 complexes were prepared similarly to other complexes using global docking followed by weak interaction analyses and local docking. These complexes were then simulated and subjected to estimation of RBFE using MM-PBSA. Obtained RBFE values were as follows: BACH1–peptide 13 complex: 201.29 ± 10.14 kcal/mol (∼17.7 kcal/mol lower than BACH2–peptide 13 complex), BCL-6–peptide 13 complex: 201.85 ± 4.07 kcal/mol (∼18.3 kcal/mol lower than BACH2–peptide 13). Although no statistically significant difference was found compared with the BACH2–peptide 13 complex, the lower RBFE values suggested that peptide 13 may be selective for the BACH2 BTB domain monomer. Among these negative controls, the BACH1 BTB domain monomer was selected for in vitro studies, as it shares 91% sequence similarity and 59% identity with the BACH2 BTB domain monomer over 138 amino acids. All RBFE values and amino acid contributions were averaged from the last 20 ns of four separate 500 ns simulations. RBFE values were reported as the mean ± standard error of the mean (SEM). t test ( p < 0.05) was used to determine statistically significant differences from PC. (B) Amino acid wise contributions to RBFE in the target BACH2 BTB domain of the protein–peptide complexes were shown. Amino acids contributing more than −3 kcal/mol were considered as significant contributors. In the target monomer of the PC BACH2 BTB domain homodimer, Met11, Tyr12, Val13, Tyr14, Ile23, Lys98, and Asn120 showed the highest contributions, with Met11-Tyr14 and Asn120 being the most dominant. In the target monomer of BACH2 BTB domain monomer–peptide 1 complex, no amino acid exceeded −3 kcal/mol. In the target monomer of BACH2 BTB domain monomer–peptide 13 complex, Glu122 and Asp123 contributed significantly, aligning with the positive control’s N terminal interaction pattern. (C) Amino acid wise contributions to RBFE in the other monomer of PC BACH2 BTB Domain Homodimer (Chain B) was shown. This monomer utilized Tyr12, Tyr14, Ile23, Leu36, Lys98, and Leu101 as determined through levels exceeding −3 kcal/mol. These contributors both aligned with the other monomer‘s contributors shown in (B), as well as validated the initial preliminary peptide prediction shown in . (D) Peptide-level amino acid contributions to RBFE are depicted for control peptides 1 and 13. Control peptide 1 contributed through Arg10 on the cell-penetrating region and Met14 and Leu27 on the designed region. Peptide 13 contributed via Arg6, Arg9, and Arg10 in the cell-penetrating region and Arg13 and Val16 in the design-exposed region. Similar to protein contributions, only amino acids with RBFE contributions of less than −3 kcal/mol were included. RBFE contributions were calculated using MM-PBSA.py with idecomp =1 to determine amino acid-level interactions.

Article Snippet: Control backbone plasmid capable of expressing only myc-DDK tag (pCMV6-Entry, mammalian vector with C-terminal Myc-DDK Tag, PS100001 ), control backbone plasmid capable of expressing only tGFP tag (pCMV6-AC-GFP Mammalian Expression Vector, PS100010 ), myc-DDK tagged BACH1 overexpression plasmid intended for use in negative control experiments (BACH1 ( NM_206866 ) Human Tagged ORF Clone-RC221628), and myc-DDK or tGFP tagged BACH2 overexpression plasmid (BACH2 ( NM_021813 ) Human Tagged ORF Clone RC214061, RG214061) were purchased from ORIGENE.

Techniques: Binding Assay, Positive Control, Control, In Vitro, Sequencing

Journal: bioRxiv

Article Title: Transient activation of potent progenitor cells is required for spinal cord regeneration

doi: 10.64898/2026.02.04.703854

Figure Lengend Snippet: (A ) Schematic representation of the genomic sequence upstream of sox2 . Grey arrowheads indicate putative binding sites for hBACH1 +/-1 mismatch. The control region marked with black arrowhead was used to detect background binding by ChIP. (B) Experimental timeline for ChIP depicting 3 daily heat shocks and SC tissue collection at either 5 or 42 dpi. (C) qRT-PCR analysis of hBACH1 enrichment at genomic regions corresponding to those marked in A at 5 and 42 dpi. Y axis represents hBach1 binding. Numbers on the X axis represent putative Bach1 binding sites from 6A, C represents a control genomic region that does not contain Bach1 binding sites. hBach1 binding was calculated using the ΔCq method and normalized to IgG immunoprecipitation (dotted line). (D) Luciferase assays using 6xBACH1 -driven Firelfy luciferase. Quantification of 6xBACH1 -Firefly luciferase after co-expression of Bach1a or Bach1b. Y axis represents that activity of Firefly luciferase normalized to control Renilla luciferase and to control wells transfected with empty pcDNA3 vector. (E) Luciferase assays using sox2 enhancer-driven Firelfy luciferase. A 402 bp genomic region containing Bach1 binding regions 5 and 6 upstream of sox2 was used. Bach1a, Bach1b with or without Maf transcription factors were co-expressed. Firefly luciferase activity was normalized to Renilla luciferase and to control wells transfected with empty pcDNA3 vector. (F-H) Experimental timeline for hBACH1 overexpression. Sox2 + cells were quantified 450 µm from the lesion at 7 and 42 dpi (G). For each section, the numbers of Sox2 + cells were normalized to the total number of nuclei. Swim endurance for hBACH1 OE fish and control siblings was performed at 42 dpi (H). (I-K) Experimental timeline for sox2 overexpression in bach1a/b mutants. Sox2 + cells were quantified 450 µm from the lesion (J). Swim endurance for sox2 OE ;bach1a/b -/- fish and bach1a/b -/- controls was performed at 42 dpi (K). Data points indicate individual animals or separate biological replicates, and sample sizes are indicated in parentheses. Two-way ANOVA with Holm-Šidák’s multiple comparisons were performed in C and G. Ordinary one-way ANOVA with Holm-Šidák’s multiple comparisons was performed in D and E. Unpaired t tests were performed in J and H. Error bars represent SEM. ns, p≥0.05; *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Immunoprecipitation was performed with either BACH1 polyclonal antibody (Proteintech #14018-1-AP) or rabbit IgG negative control antibody (Cell Signaling Technology #2729).

Techniques: Sequencing, Binding Assay, Control, Quantitative RT-PCR, Immunoprecipitation, Luciferase, Expressing, Activity Assay, Transfection, Plasmid Preparation, Over Expression