p75ntr plasmids  (Addgene inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: p75NTR promotes tooth rhythmic mineralization via upregulation of BMAL1/CLOCK

doi: 10.3389/fcell.2023.1283878

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: BMAL1 expression plasmid, CLOCK expression plasmid, firefly luciferase reporter plasmid containing the p75NTR promoter, and the corresponding negative control plasmid were purchased from GeneChem (Shanghai, China).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: p75NTR promotes tooth rhythmic mineralization via upregulation of BMAL1/CLOCK

doi: 10.3389/fcell.2023.1283878

Figure Lengend Snippet: p75NTR , Bmal1 , and Clock followed similar circadian rhythms in vitro and in vivo . (A) After synchronization using dexamethasone, ectomesenchymal stem cells (EMSCs) were harvested at the indicated time point and analyzed using qRT-PCR. (B) Tooth germs were dissected from postnatal day 1 wildtype mice every 4 h starting at 8:00 a.m. The mRNA expression levels of Bmal1 , Clock , Per1 , Per2 , Cry1 , and p75NTR in the tooth germ were analyzed using qRT-PCR at the indicated time points. Data are shown as the means ± standard deviation of three independent experiments (n = 3).

Article Snippet: BMAL1 expression plasmid, CLOCK expression plasmid, firefly luciferase reporter plasmid containing the p75NTR promoter, and the corresponding negative control plasmid were purchased from GeneChem (Shanghai, China).

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: p75NTR promotes tooth rhythmic mineralization via upregulation of BMAL1/CLOCK

doi: 10.3389/fcell.2023.1283878

Figure Lengend Snippet: p75NTR positively regulated the expression of BMAL1 and CLOCK. (A) Representative western blots and quantitative analysis of BMAL1 and CLOCK proteins in OE-p75NTR and OE-NC ectomesenchymal stem cells (EMSCs) (n = 3). (B) Representative Western blot analysis and quantification of BMAL1 and CLOCK in the tooth germs of wildtype (WT) and knockout (KO) mice (n = 3). (C) Immunofluorescence staining of p75NTR, BMAL1, and CLOCK in OE-NC and OE-p75NTR EMSCs. Scale bar = 50 μm. Data are shown as mean ± standard deviation. ** p ≤ 0.01 and **** p ≤ 0.0001.

Article Snippet: BMAL1 expression plasmid, CLOCK expression plasmid, firefly luciferase reporter plasmid containing the p75NTR promoter, and the corresponding negative control plasmid were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Western Blot, Knock-Out, Immunofluorescence, Staining, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: p75NTR promotes tooth rhythmic mineralization via upregulation of BMAL1/CLOCK

doi: 10.3389/fcell.2023.1283878

Figure Lengend Snippet: p75NTR positively regulated the odontogenic differentiation and mineralization of EMSCs by upregulating the expression of BMAL1/CLOCK. (A, B) EMSCs stably overexpressing BMAL1 or CLOCK (OE-BMAL1 or OE-CLOCK) were generated via lentiviral transduction and treated with the mineralization-inducing medium. (A) ALP staining was performed on day 7 and (B) alizarin red staining was performed on day 21 (n = 3). Scale bar = 300 μm. (C) Representative Western blot and quantification of DMP1, DSPP, RUNX2, and ALP in EMSCs (n = 3). (D) EMSCs overexpressing p75NTR were transfected with BMAL1 and CLOCK siRNAs (OE-p75NTR + si-BMAL1/CLOCK) or negative control siRNA (OE-p75NTR + si-NC). These EMSCs were treated with the mineralization-inducing medium. ALP and alizarin red staining were performed on days 7 and 21, respectively (n = 3). Data are shown as mean ± standard deviation. ** p ≤ 0.01, *** p ≤ 0.005, and ****p ≤ 0.0001. EMSCs, ectomesenchymal stem cells.

Article Snippet: BMAL1 expression plasmid, CLOCK expression plasmid, firefly luciferase reporter plasmid containing the p75NTR promoter, and the corresponding negative control plasmid were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Stable Transfection, Generated, Transduction, Staining, Western Blot, Transfection, Negative Control, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: p75NTR promotes tooth rhythmic mineralization via upregulation of BMAL1/CLOCK

doi: 10.3389/fcell.2023.1283878

Figure Lengend Snippet: BMAL1/CLOCK inhibited p75NTR promoter activity and protein expression. (A–D) The mRNA and protein levels of p75NTR in ectomesenchymal stem cells (EMSCs) overexpressing BMAL1 or CLOCK were detected using (A and C) qRT-PCR and (B and D) Western blot analyses (n = 3). (E) DNA-binding motifs of BMAL1 and CLOCK were obtained from the JASPAR database. (F) Predicted BMAL1/CLOCK binding sites in the p75NTR promoter region according to the JASPAR database. (G) Dual-luciferase reporter assay was performed to measure the activities of the wildtype p75NTR promoter in HEK293T cells transfected with BMAL1 expression plasmids and/or CLOCK expression plasmids (n = 3). Data are shown as the mean ± standard deviation. ** p ≤ 0.01, *** p ≤ 0.005, and **** p ≤ 0.0001.

Article Snippet: BMAL1 expression plasmid, CLOCK expression plasmid, firefly luciferase reporter plasmid containing the p75NTR promoter, and the corresponding negative control plasmid were purchased from GeneChem (Shanghai, China).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Luciferase, Reporter Assay, Transfection, Standard Deviation

Journal: ACS chemical neuroscience

Article Title: Small molecules targeting PTPσ—Trk interactions promote sympathetic nerve regeneration

doi: 10.1021/acschemneuro.1c00854

Figure Lengend Snippet: Representative western blots (left) probing for PTPσ and TrkA following TrkA-RFP immunoprecipitation. HEK-293t cells were transfected with TrkA-RFP and PTPσ, treated with either vehicle (DMSO), (A) HJ-01, (B) HJ-02, or (C) HJ-03 then stimulated with CSPGs and NGF. Quantification (right) of PTPσ that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. HJ-01 and HJ-02 reduced the amount of PTPσ that bound to TrkA-RFP at 1μM, but HJ-03 had no effect. (D) HEK-293t cells were transfected with TrkA-RFP and p75NTR, treated with vehicle (DMSO) and HJ-02, then stimulated with CSPGs and NGF. Quantification (right) of p75NTR that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. Data are mean ± SD, n=5-9 experiments; n.s.- not significant, *p<.05, **p<.01 vs. vehicle treated control, one-way ANOVA (Dunnett multiple comparisons post-test).

Article Snippet: We would like to thank Dr. Moses Chao (Skirball Institute, NYU) for the HEK-TrkB cells and for the TrkA-RFP and p75NTR plasmids (Addgene plasmid # 24093, 24091).

Techniques: Western Blot, Immunoprecipitation, Transfection

Journal: ACS chemical neuroscience

Article Title: Small molecules targeting PTPσ—Trk interactions promote sympathetic nerve regeneration

doi: 10.1021/acschemneuro.1c00854

Figure Lengend Snippet: Representative western blots (left) probing for PTPσ and TrkA following TrkA-RFP immunoprecipitation. HEK-293t cells were transfected with TrkA-RFP and PTPσ, treated with either vehicle (DMSO), (A) HJ-01, (B) HJ-02, or (C) HJ-03 then stimulated with CSPGs and NGF. Quantification (right) of PTPσ that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. HJ-01 and HJ-02 reduced the amount of PTPσ that bound to TrkA-RFP at 1μM, but HJ-03 had no effect. (D) HEK-293t cells were transfected with TrkA-RFP and p75NTR, treated with vehicle (DMSO) and HJ-02, then stimulated with CSPGs and NGF. Quantification (right) of p75NTR that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. Data are mean ± SD, n=5-9 experiments; n.s.- not significant, *p<.05, **p<.01 vs. vehicle treated control, one-way ANOVA (Dunnett multiple comparisons post-test).

Article Snippet: To test the hypothesis that small molecules HJ-01 and HJ-02 disrupt the binding of TrkA and PTPσ, HEK-293t cells were transfected with full length PTPσ or p75NTR (Addgene plasmid # 24091) and TrkA-RFP plasmid (Addgene plasmid #24093), and pulled down using RFP-trap nanobody conjugated to magnetic agarose (Chromotek).

Techniques: Western Blot, Immunoprecipitation, Transfection

Journal: ACS chemical neuroscience

Article Title: Small molecules targeting PTPσ—Trk interactions promote sympathetic nerve regeneration

doi: 10.1021/acschemneuro.1c00854

Figure Lengend Snippet: Representative western blots (left) probing for PTPσ and TrkA following TrkA-RFP immunoprecipitation. HEK-293t cells were transfected with TrkA-RFP and PTPσ, treated with either vehicle (DMSO), (A) HJ-01, (B) HJ-02, or (C) HJ-03 then stimulated with CSPGs and NGF. Quantification (right) of PTPσ that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. HJ-01 and HJ-02 reduced the amount of PTPσ that bound to TrkA-RFP at 1μM, but HJ-03 had no effect. (D) HEK-293t cells were transfected with TrkA-RFP and p75NTR, treated with vehicle (DMSO) and HJ-02, then stimulated with CSPGs and NGF. Quantification (right) of p75NTR that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. Data are mean ± SD, n=5-9 experiments; n.s.- not significant, *p<.05, **p<.01 vs. vehicle treated control, one-way ANOVA (Dunnett multiple comparisons post-test).

Article Snippet: We would like to thank Dr. Moses Chao (Skirball Institute, NYU) for the HEK-TrkB cells and for the TrkA-RFP and p75NTR plasmids (Addgene plasmid # 24093, 24091).

Techniques: Western Blot, Immunoprecipitation, Transfection, Control