Journal: The FASEB Journal

Article Title: MyD88‐mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms

doi: 10.1096/fj.202100777rr

Figure Lengend Snippet: FIGURE 4 Targeted ablation of MyD88 inhibits autophagy and FOXO transcription factors in denervated skeletal muscle. Control (sham) and 5d- or 14d-denervated GA or TA muscle of MyD88f/f and MyD88myoKO mice were analyzed by performing QRT-PCR assay and western blot. A, Relative mRNA levels of autophagy-related molecules, LC3B, Beclin1, ATG5, ATG12, and GABARAPL1 in sham-operated and 5d-denervated muscle of MyD88f/f and MyD88myoKO mice. B, Relative mRNA levels of LC3B, Beclin1, ATG5, ATG12, and GABARAPL1 in sham-operated and 14d-denervated muscle of MyD88f/f and MyD88myoKO mice. C, Representative immunoblots showing protein levels of LC3BI and LC3BII and unrelated protein GAPDH. D, Densitometry quantification of LC3BII levels in sham-operated and denervated GA of MyD88f/f and MyD88myoKO mice. E, Representative immunoblots demonstrating levels of phosphorylated and total FoxO1, FoxO3, and FoxO4 proteins in sham-operated and 5d-denerated TA muscle of MyD88f/f and MyD88myoKO mice. F, Densitometry quantification of levels of FOXO proteins in sham-operated and denervated TA muscle of MyD88f/f and MyD88myoKO mice. n = 3-6 mice per group. Data are presented as mean ± SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. *P < .05 (values significantly different from contralateral sham- operated TA muscle of MyD88f/f or MyD88myoKO mice). #P < .05 (values significantly different from denervated muscle of MyD88f/f mice). D, Denervated; S, sham

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 4 of 17 | PARVEEN Et Al. Biotechnology, sc- 7221), anti- phospho- FoxO1 (Thr24)/ FoxO3a (Thr32) (Cell Signaling Technology #9464), antiFoxO1 (Cell Signaling Technology #2880, anti- FoxO3a (Cell Signaling Technology #12829), anti- phospho- FoxO4 Cell Signaling Technology #9471) anti- FoxO4 (Cell Signaling Technology #2499), anti- LC3BI/II (Cell Signaling Technology, #2775), anti- Fn14 (Cell Signaling technology, #4403), anti- phospho- p65 (Cell Signaling Technology, #3033), anti- p65 (Cell Signaling Technology, #8242), antip100/52 (Cell Signaling Technology, #4882) anti- p- AMPK (Cell Signaling Technology, #2535), anti- AMPK (Cell Signaling Technology, #2532), anti- phospho- mTOR (Cell Signaling Technology, #2971), anti- mTOR (Cell Signaling Technology, #2972), anti- PERK (Cell Signaling technology, #5683), anti- ATF4 (Cell Signaling Technology, #11815), anti- CHOP (Cell Signaling Technology #2895), anti- IRE1α (Cell Signaling Technology), anti- sXBP1 (Cell Signaling Technology #12782), and anti- GAPDH (Cell Signaling Technology, #2118).

Techniques: Control, Quantitative RT-PCR, Western Blot, Comparison

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Preparation and characterization of engineered ADGRG1-targeting and hypoxia-treated EVs. (A)Induced fit docking analysis of ADGRG1-binding peptide (A1TP, 7 peptides) and extracellular domain of ADGRG1 protein (PDB database: 7SF8). (B) Analysis of the binding of the A1TP to purified ADGRG1 proteins using a microscale thermophoresis (MST) binding assay. (C) Induced fit docking analysis of A1TP-PEG and extracellular domain of ADGRG1 protein. (D) The binding free energy between A1TP or A1TP-PEG and ADGRG1 were calculated using molecular dynamics simulations. Lower values indicate more stable interactions, with values less than or equal to −20 considered as stable binding modes. (E) Schematic illustration of the conjugating reaction between DSPE-PEG-Alkyne and A1TP. Schematic illustration of the fabrication of A1TP-HX-EVs through external modification by A1TP anchoring. Specific steps for the synthesis of DSPE-PEG-A1TP (DPA) are shown in . (F) FT-IR analysis showed the characteristic peaks of the DSPE-PEG-A1TP. The new triazole ring itself showed a characteristic C=N stretching vibration, a peak at 1538 cm −1 revealed the successful conjugation of A1TP. (G) H Nuclear magnetic resonance (NMR) spectra of DSPE-PEG-A1TP in D2O. The hydrogen signatures of the phenyl and phenol groups at 7.5-8.0 ppm confirmed the successful conjugation of DSPE to A1TP. (H) Western blot analysis verified the presence of three EV marker proteins (ALIX, TSG101, and CD81) and one EV negative marker (GM130) in EVs, HX-EVs, and A1TP-HX-EVs. (I) Transmission electron microscopy (TEM) images of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 200 nm. (J) Zeta potentials of EVs, HX-EVs and A1TP-HX-EVs, n = 3. Two-tailed unpaired Student's t-test was used for statistical analysis. ns, not significant. A two-tailed unpaired Student's t-test was used for statistical analysis. (K) Representative images of the spherical morphology and dispersion states of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 500 nm. (L) Size distributions of EVs, HX-EVs and A1TP-HX-EVs.

Article Snippet: Finally, the presence of the characteristic EV markers Alix (92880, Cell Signaling Technology), CD81 (56039, Cell Signaling Technology) and TSG101 (sc-7964, Santa Cruz Biotechnology) was confirmed by Western blot analysis.

Techniques: Binding Assay, Purification, Microscale Thermophoresis, Modification, Conjugation Assay, Nuclear Magnetic Resonance, Western Blot, Marker, Transmission Assay, Electron Microscopy, Two Tailed Test, Dispersion

Journal: mSphere

Article Title: The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells

doi: 10.1128/msphere.00056-23

Figure Lengend Snippet: Proteomics analyses in 38HK identify the E2F4/p130 complex as a partner of ΔNp73α. (A) Illustration of the proteomics approach used to identify nuclear binding partners of ΔNp73α. (B) Expression and purification of ΔNp73α-TAP complexes. (B, upper panel) Distribution of ΔNp73α-TAP in cytoplasmic and nuclear fractions of 38HK extracts. The 2 fractions were centrifuged and analyzed by Western blotting using an anti-TAP antibody. T, total extract; S, supernatant; P, pellet. ΔNp73α-TAP is present mainly in the nuclear fraction (Cyt[P] and Nuc[S], see also Materials and Methods section). (B, lower panel) Silver-stained 10% SDS-PAGE analysis of elution fractions 1 to 5 from the second affinity purification step (calmodulin resin) for ΔNp73α-TAP and control (TAP) purifications. An additional elution with SDS was performed to recover all the remaining proteins. (C) Pathway analysis of nuclear ΔNp73α binding partners using the Reactome database . Only significant pathways are shown (defined by a false discovery rate [FDR] value ≤ 0.02) and ranked based on the -log 10 of the associated P value (*: ≤ 0.05; **: ≤ 0.01; ***: ≤ 0.001; ****: ≤ 0.0001). Histogram bar size shows the number of input proteins involved in the corresponding pathway. See also S1 Table. (D) Sucrose gradient/co-IP experiments on endogenous 38HK proteins. (D, upper panel) Sucrose fractions of 38HK nuclear extracts stably expressing HA-ΔNp73α were migrated on a 10% SDS-PAGE gel, and analyzed by Western blotting using antibodies recognizing the HA tag, E2F4, and p130 proteins. (D, lower panel) Indicated fractions were immunoprecipitated using anti-HA conjugated beads. Unfractionated nuclear extract (NE) was loaded on the same gel as a reference for protein migration. Images for NE and sucrose fractions are derived from different exposures of the same membrane. See also original Western blot image on Mendeley data.

Article Snippet: Western blot detection of endogenous proteins was performed using the following antibodies: p73 (Abcam, ref: ab215038), E2F4 (Santa Cruz Biotechnology, ref. sc-398543X), E2F5 (Genetex, ref. GTX129491), DP1 (Abcam, ref. ab124678), p130 (Cell Signaling, ref. 13610S) β-actin (clone C4, MP Biomedicals), and GAPDH (6C5, ref. sc-32233, Santa Cruz).

Techniques: Binding Assay, Expressing, Purification, Western Blot, Staining, SDS Page, Affinity Purification, Control, Co-Immunoprecipitation Assay, Stable Transfection, Immunoprecipitation, Migration, Derivative Assay, Membrane

Journal: mSphere

Article Title: The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells

doi: 10.1128/msphere.00056-23

Figure Lengend Snippet: ΔNp73α establishes a direct PPI with E2F4. (A, left panel) Representative data set for GPCA analysis of binary interactions between ΔNp73/TAp63 proteins and components of the E2F4/p130 complex. Pairwise combinations of Gluc1-ΔNp73/TAp63 and Gluc2-E2F4/DP1/p130 fusion constructs were co-transfected in HEK293T cells. After 48 h, the luciferase activity was measured and expressed as normalized luminescence ratio (NLR) . The interactions of Gluc1-E2F4 with Gluc2-E2F4/DP1/p130 proteins are reported as internal controls. Error bars show standard deviations derived from triplicate measurements. (A, right panel) 8% SDS-PAGE analysis of the expression of Gluc1-fused ΔNp73/TAp63 and E2F4 proteins, and of Gluc2-fused E2F4, DP1 and p130 proteins in HEK293T cells. Proteins were visualized by Western blotting using an anti-Gluc antibody, which allows for detection of both Gluc1 and Gluc2 fragments, albeit with lower efficiency for Gluc2. The multiple bands detected for E2F4 likely correspond to multiple phosphorylation states of the protein as shown in . (B) Expression of ΔNp73α, E2F4-5, DP1, and p130 proteins in 38HK cells transfected with scramble (SC) siRNAs or siRNAs against either E2F4 (siE2F4), E2F5 (siE2F5), or E2F4 plus E2F5 (siE2F4-5). (B, left panel) mRNA levels measured by RT-qPCR. Error bars represent standard deviations derived from three independent experiments. (B, right panel) Protein levels as determined by Western blotting analysis. All samples were migrated on the same gel. See original Western blot image on Mendeley data. (C) Sucrose gradient/co-IP experiments using nuclear extracts from 38HK in the scramble (C, upper panels) and siE2F4-5 (C, lower panels) conditions. Indicated fractions were immunoprecipitated by anti-HA beads. HA-ΔNp73α, E2F4, E2F5, DP1, and p130 were detected by Western blotting. IgG(H) chains migrate in close proximity with E2F5 and DP1 proteins. NE, unfractionated nuclear extract. See also Fig. S2A and for experiments under single siE2F4 or siE2F5 conditions. See also legend of Fig. 1D . (D) Quantification of immunoprecipitated HA-ΔNp73α DP1 and p130 proteins in the scramble and siE2F4-5 conditions of the co-IP experiments shown in panel (C). For each protein, band intensities in the individual fractions are normalized to the intensity of the corresponding band in the NE control (I fr /I NE ). Then, the I fr /I NE values of fractions 12 to 16 are summed to get an estimate of the total protein levels. The total DP1 and p130 protein values are additionally normalized to HA-ΔNp73α levels in the corresponding condition.

Article Snippet: Western blot detection of endogenous proteins was performed using the following antibodies: p73 (Abcam, ref: ab215038), E2F4 (Santa Cruz Biotechnology, ref. sc-398543X), E2F5 (Genetex, ref. GTX129491), DP1 (Abcam, ref. ab124678), p130 (Cell Signaling, ref. 13610S) β-actin (clone C4, MP Biomedicals), and GAPDH (6C5, ref. sc-32233, Santa Cruz).

Techniques: Construct, Transfection, Luciferase, Activity Assay, Derivative Assay, SDS Page, Expressing, Western Blot, Phospho-proteomics, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Immunoprecipitation, Control

Journal: mSphere

Article Title: The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells

doi: 10.1128/msphere.00056-23

Figure Lengend Snippet: Identification of domains/regions mediating the ΔNp73α-E2F4 interaction. (A, left panel) Schematic representation of the Gluc1-fused TA/ΔNp73α and Gluc2-fused E2F4 constructs analyzed by GPCA. The ΔN(1 to 433) construct corresponds to the ΔNp73β isoform. Domains of TAp73α/ΔNp73α: transactivation domain (TAD, yellow); DNA-binding domain (DBD, blue); oligomerization domain (OD, red); sterile alpha motif domain (SAM, green); 13-amino-acid peptide specific for ΔNp73 isoforms (ΔN peptide, purple). Domains of E2F4: DNA-binding domain (DBD, blue); DP-binding and dimerization domain (DP-dim, red); transactivation domain (TAD, yellow); pocket protein binding domain (PPBD, orange). (A, right panel) Expression levels of Gluc1- and Gluc2-fused TA/ΔNp73α and E2F4 constructs in HEK293T cells. Proteins were resolved on a 25%/10% gradient SDS-gel and visualized by Western blotting using an anti-Gluc antibody. (B) GPCA analysis of the pairwise interactions of Gluc1-TAp73α or Gluc1-ΔNp73α FL with Gluc2-E2F4 FL. (C) MBP-pulldown analysis of the interactions of TAp73α and ΔNp73α FL with E2F4 FL. Amylose resin coupled to MBP-TAp73α or MBP-ΔNp73α FL constructs was incubated with clarified extracts from HEK293T cells transiently expressing 3xFlag-E2F4 FL. PD reactions were migrated on 2 separate 10% SDS-PAGE gels. One gel was used for E2F4 detection by Western blotting using an anti-Flag antibody, the second gel for detection of MBP fusions by Coomassie staining. The negative control MBP-SAM construct (residues 405 to 502 of ΔNp73α) was migrated on the same gel (see original Western blot images on Mendeley data). (D) GPCA analyses of pairwise ΔNp73α/E2F4 interactions. (D, left panel) Gluc1-ΔNp73α deletion constructs versus Gluc2-E2F4 FL. (D, right panel) Gluc1-ΔNp73α FL versus Gluc2-E2F4 deletion constructs. (B and D) The NLR values were normalized to the ΔNp73α-E2F4 FL interaction and are averages from 3 independent experiments (with each experiment being performed in triplicate). P values are obtained from unpaired t test, n = 3 biological triplicates (*: P < 0.05; **: P < 0.01). (E) Interaction analyses using recombinant purified proteins. (E, left panel) Schematic representation of the constructs used for the minimal E2F4 s /DP1 s heterodimer. (E, right panel) Pulldown (PD) experiment using MBP-ΔN(1 to 433) (i.e., -ΔNp73β) and MBP-ΔN(1 to 348) or MBP (negative control) proteins and the minimal E2F4 s /DP1 s heterodimer. PD samples were migrated on 2 separate 10% SDS-PAGE gels. One gel was used for E2F4 s /DP1 s detection by Western blotting using anti-His and anti GST antibodies, and the second gel for detection of MBP-ΔNp73 fusions by Coomassie staining. (F) Proposed mechanism of ΔNp73α interaction with the E2F4/p130 complex. Red hatched box: E2F4 binding site.

Article Snippet: Western blot detection of endogenous proteins was performed using the following antibodies: p73 (Abcam, ref: ab215038), E2F4 (Santa Cruz Biotechnology, ref. sc-398543X), E2F5 (Genetex, ref. GTX129491), DP1 (Abcam, ref. ab124678), p130 (Cell Signaling, ref. 13610S) β-actin (clone C4, MP Biomedicals), and GAPDH (6C5, ref. sc-32233, Santa Cruz).

Techniques: Construct, Binding Assay, Sterility, Protein Binding, Expressing, SDS-Gel, Western Blot, Incubation, SDS Page, Staining, Negative Control, Recombinant, Purification

Journal: mSphere

Article Title: The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells

doi: 10.1128/msphere.00056-23

Figure Lengend Snippet: Expression and regulation of ΔNp73α-E2F4/p130 complex components in primary HPK and transformed 38HK cells. (A, left panel) Immunoblotting of ΔNp73α, E2F4, and p130 proteins in human foreskin HPK and 38HK cell lines from 3 different donors (D1 to D3). *: HPK(D2) and 38HK(D2) are from the same donor. 38HK(D2) is an early passage (i.e., passage 16) cell line, whereas the 38HK(D3) is a late passage cell line, which was used throughout this study. (A, right panel) Phosphorylation of p130. Total lysates of HPK and 38HK cells were incubated in presence or absence of λ-phosphatase (λ-pp) (8 U/μL). Note the small migration shift between the λ-pp- and λ-pp+ conditions. (B, left panel) Quantification of protein bands from the Western blot analyses shown in panel (A), left. (B, right panel) mRNA levels of the p130 gene in the different cell lines as measured by RT-qPCR. *: P < 0.05. (C) Distribution of ΔNp73α and E2F4 and p130 proteins in the nuclear soluble (Nuclear[S]), chromatin, and cytoplasm fractions of 38HK. (D, left panel) Representative data set of the GPCA analysis of the pairwise interactions between Gluc1-38.E7/E6 or Gluc1-16.E7 proteins and Gluc2-fused RB proteins co-expressed in HEK293T cells. Error bars show standard deviations derived from triplicate measurements. (D, right panels) Expression levels of Gluc1-38.E7/E6 and Gluc1-16.E7 proteins and Gluc2-RB proteins in HEK293T cells. Gluc1-38.E7/E6 and Gluc1-16.E7 were migrated on a 12% SDS-PAGE gel, whereas Gluc2-RB proteins on a 8% gel. (E) Kinetics of p130 degradation in different keratinocyte cell lines. (E, left panels) Western blot analyses of total lysates of 38HK, 16HK, and primary HPKs stably transfected with pLXSN plasmid. All cells were treated with cycloheximide (10 μg/mL) during 12 h. Cells were collected at the indicated time points. (E, right panel) p130 levels in the three cell lines at the different time points. The data reported are normalized to actin and to the values at time = 0 (100%). Error bars represent standard deviation values derived from 3 (16HK and 38HK) or 2 (HPK + pLXSN) independent experiments. P values (*: P < 0.05) refer to the differences between 38HK and 16HK at 2 and 4 h, and are calculated using the unpaired t test, n = 3 biological triplicates.

Article Snippet: Western blot detection of endogenous proteins was performed using the following antibodies: p73 (Abcam, ref: ab215038), E2F4 (Santa Cruz Biotechnology, ref. sc-398543X), E2F5 (Genetex, ref. GTX129491), DP1 (Abcam, ref. ab124678), p130 (Cell Signaling, ref. 13610S) β-actin (clone C4, MP Biomedicals), and GAPDH (6C5, ref. sc-32233, Santa Cruz).

Techniques: Expressing, Transformation Assay, Western Blot, Phospho-proteomics, Incubation, Migration, Quantitative RT-PCR, Derivative Assay, SDS Page, Stable Transfection, Transfection, Plasmid Preparation, Standard Deviation

Journal: mSphere

Article Title: The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells

doi: 10.1128/msphere.00056-23

Figure Lengend Snippet: ΔNp73α cooperates with E2F4/p130 in HPV-negative cancer cells. (A) Sucrose gradient/co-IP experiments using nuclear extracts of HNC-136 cells. Sucrose fractions were immunoprecipitated using anti-p73 antibody-coupled beads and analyzed for endogenous ΔNp73α, E2F4, E2F5, and p130 proteins. Note that the p130 signal (highlighted by a red asterix) is partially masked by closely migrating nonspecific band. See also expanded Western blot images on Mendeley data. See also legend of Fig. 1D . (B) Expression profiles of selected genes in CAL-51 cells. mRNA levels determined by RT-qPCR under conditions of E2F4-5 (white histograms) or ΔNp73α (gray histograms) depletion. The red line refers to the mRNA level in the SC or S conditions (set to 1). “siE2F4-5/SC” and “AS/S ΔNp73α” values > 1 and < 1 indicate upregulation and downregulation of gene expression, respectively. The data are averages from 3 independent experiments, with error bars representing standard deviation values. P values are obtained from unpaired t test, n = 3 biological triplicates (*: P < 0.05; **: P < 0.01; ***: P < 0.001). See also Fig. S4D to for E2F4-5, and ΔNp73α levels in the scramble and knockdown conditions.

Article Snippet: Western blot detection of endogenous proteins was performed using the following antibodies: p73 (Abcam, ref: ab215038), E2F4 (Santa Cruz Biotechnology, ref. sc-398543X), E2F5 (Genetex, ref. GTX129491), DP1 (Abcam, ref. ab124678), p130 (Cell Signaling, ref. 13610S) β-actin (clone C4, MP Biomedicals), and GAPDH (6C5, ref. sc-32233, Santa Cruz).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Expressing, Quantitative RT-PCR, Gene Expression, Standard Deviation, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: AMPA and NMDA Receptors in Hippocampus of Rats with Fluoride-Induced Cognitive Decline

doi: 10.3390/ijms252111796

Figure Lengend Snippet: mRNA and protein levels of GluN1 subunits of NMDARs in hippocampal cells. ( A ) Relative expression of Grin1 gene in the hippocampus of rats consumed different F - doses normalized to that of a pair of reference genes ( Eef1a+Ppia ). Average values ± SEM (n = 10). ( B , C ) Protein expression of native and phosphorylated (Ser890) forms of GluN1 subunit in cytosolic and membrane fractions, respectively. Shown are typical immunoblots and average values ± SEM (n = 8).

Article Snippet: Rabbit antibodies to GluA1 (#13185), phospho-GluA1 (#8084), GluA2 (#5306), phospho-GluA2 (#3921), GluA3 (#4676), GluN1 (#5704), phospho-GluN1 (#3381), GluN2A (#4205), phospho-GluN2A (#4206), GluN2B (#4207) and phospho-GluN2B (#4208) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Membrane, Western Blot