Journal:

Article Title: CD40 is expressed and functional on neuronal cells

doi: 10.1093/emboj/21.4.643

Figure Lengend Snippet: Fig. 4. CD40 ligation rescues neuronal cell injury and inhibits JNK activation induced by NGF-β or serum withdrawal. (A) Western blotting (top) shows expression of Bcl-xL and Bax in wild-type (left) or CD40-deficient (right) primary cultured neurons that were incubated with serum (control) or were subjected to serum withdrawal (SW) in the presence or absence of CD40L for 12 h. Graph (bottom) summarizes band density ratios of Bcl-xL:Bax (mean ± 1 SEM) for the above, with n = 3 for each condition presented. (A, left) Significant differences were noted between incubation of serum (control) in the presence or absence of CD40L (p <0.05), as well as between cells subjected to SW in the presence or absence of CD40L (p <0.001). (A, right) No significant between-group differences were found (p >0.05). (B) LDH release assay (mean ± 1 SEM, n = 3 for each condition presented) shows that CD40 ligation significantly (p <0.001) reduces LDH release in primary neuronal cells at 72 and 96 h post-withdrawal of serum (left), but no CD40L effect was noted in CD40-deficient primary neurons (right) (p >0.05). (C) Western blotting (top) shows phosphorylated JNK1 (p46) in differentiated PC12 cells after NGF-β withdrawal in the presence (right) or absence (left) of CD40L for 30, 60 or 90 min as indicated. Graph (bottom) summarizes band density ratios (mean ± 1 SEM) of phospho-JNK1:total JNK1 for the above with n = 3 for each condition presented. Significant differences were found when comparing ‘before’ with ‘after’ CD40L ligation at 30 or 60 min (p <0.01). (D) Immune complex kinase assay (top) shows phosphorylation of c-Jun fusion protein before (left) or after (right) CD40 ligation of differentiated PC12 cells, which were treated as described in (C). Graph (bottom) summarizes band densities (mean ± 1 SEM) for the above, with n = 3 for each condition presented. Significant differences were found when comparing ‘before’ with ‘after’ CD40L ligation at 30 or 60 min (p <0.001). (E and F) Western blotting (top) shows phosphorylated JNK1 (p46) in wild-type (E) or CD40 deficient (F) primary neuronal cells after serum withdrawal in the presence (right) or absence (left) of CD40L for the time points indicated. Graph (bottom) summarizes band density ratios (mean ± 1 SEM) of phospho-JNK1:total JNK1 for the above with n = 3 for each condition presented. For (E), significant differences were found when comparing ‘before’ with ‘after’ CD40L ligation at each time point (p <0.05). For (F), no significant between-groups differences were found (p >0.05). (G and H) Immune complex kinase assay (top) shows phosphorylation of c-Jun fusion protein before (left) or after (right) CD40 ligation of wild-type (G) or CD40-deficient (H) primary neurons that were treated as described in (E and F). Graph (bottom) summarizes band densities (mean ± 1 SEM) for the above, with n = 3 for each condition presented. For (G), significant differences were found when comparing ‘before’ with ‘after’ CD40L ligation at 30 or 60 min (p <0.01). For (H), no significant between-groups differences were noted (p >0.05).

Article Snippet: JNK phosphorylation and activity were determined using the SAPK/JNK assay kit (Cell Signaling Technology), using antibodies against total or phospho-JNK1 or phospho-c-Jun.

Techniques: Ligation, Activation Assay, Western Blot, Expressing, Cell Culture, Incubation, Lactate Dehydrogenase Assay, Immune Complex Kinase Assay

Journal: eNeuro

Article Title: Evidence That Dmrta2 Acts through Repression ofPax6in Cortical Patterning and Identification of a Mutation Impairing DNA Recognition Associated with Microcephaly in Human

doi: 10.1523/eneuro.0377-24.2025

Figure Lengend Snippet: Figure 5. RIME-purified Dmrta2 complexes isolated from dissected cortical tissue contain multiple subunits of NuRD and the NuRD interacting Zfp423 protein. A, Top panels, RT-qPCR using cDNA prepared from dissected cortices of E12.5 embryos with primers flanking the inserted 2× HA sequence, showing that the tagged allele is detectable in the two F0 Dmrta22XHA mice (clones 1 and 2) and is absent in WT mice. A Western blot is also shown dem- onstrating that the HA-tagged protein is expressed in Dmrta22xHA mice. Bottom panels, immunostainings showing that the HA-tagged allele is expressed similarly to Dmrta2 in the telencephalon. h, hem; cp, choroid plexus; dtel, dorsal telencephalon; e, eye; et, eminentia thalami; vtel, ventral telencephalon.

Article Snippet: Then the membrane was blocked with Tris-buffered saline (TBS) with 5% non-fat milk for 1 h, at RT, and incubated at 4°C, O/N, with the following primary antibodies: rabbit anti-Flag (1:1,000); rabbit anti-HA (1:1,000); rabbit anti-H3 (1:50,000, Millipore, catalog #07-690); mouse anti-Myc (1:1,000, Sigma, catalog #M4439); mouse anti-Zfp423 (1:1,000); and the NurD complex antibody kit (Cell Signaling, catalog #8349T).

Techniques: Isolation, Quantitative RT-PCR, Sequencing, Clone Assay, Western Blot

Journal: eNeuro

Article Title: Evidence That Dmrta2 Acts through Repression ofPax6in Cortical Patterning and Identification of a Mutation Impairing DNA Recognition Associated with Microcephaly in Human

doi: 10.1523/eneuro.0377-24.2025

Figure Lengend Snippet: Figure 6. The Dmrta2-Pax6 interaction requires the recruitment of the HDAC-NuRD complex by Zfp423. A, B, Coimmunoprecipitation assays as indicated with Zfp423, Flag-Dmrta2, and a Flag-Dmrta2 mutant lacking the DM domain (Flag-Dmrta2 ΔDM) overexpressed in HEK293T cells as indicated. Note in A that Flag-Dmrta2 pulled down Zpf423 (line 1–2) and that, conversely, Zpf423 coprecipitated Flag-Dmrta2 (line 3–4). Note in B that Flag-Dmrta2 ΔDM does not pull down Zfp423 (line 4). n = 3. Segments from the same blot have been spliced together to show side by side the results of the WT and the ΔDM Dmrta2 mutant. C, Full-length mouse Myc-tagged Zfp423 was synthesized in vitro and incubated with GST alone or with GST fusion proteins bound to

Article Snippet: Then the membrane was blocked with Tris-buffered saline (TBS) with 5% non-fat milk for 1 h, at RT, and incubated at 4°C, O/N, with the following primary antibodies: rabbit anti-Flag (1:1,000); rabbit anti-HA (1:1,000); rabbit anti-H3 (1:50,000, Millipore, catalog #07-690); mouse anti-Myc (1:1,000, Sigma, catalog #M4439); mouse anti-Zfp423 (1:1,000); and the NurD complex antibody kit (Cell Signaling, catalog #8349T).

Techniques: Mutagenesis, Synthesized, In Vitro, Incubation

Journal: eNeuro

Article Title: Evidence That Dmrta2 Acts through Repression ofPax6in Cortical Patterning and Identification of a Mutation Impairing DNA Recognition Associated with Microcephaly in Human

doi: 10.1523/eneuro.0377-24.2025

Figure Lengend Snippet: Figure 7. Model illustrating the action of Dmrta2 and related family members in cortical patterning. Our results provide experimental evidence showing that downstream of hem derived Wnt signals, Dmrta2 acts through the repression of Pax6 to promote caudomedial fate, and it cooperates with Pax6 to repress ventral determinants such as Gsx1 and Gsx2, extending previous studies having defined Pax6 regulation by Dmrt factors (Konno et al., 2012; Saulnier et al., 2013; Young et al., 2017). In accordance with Konno et al. (2019), the data suggest that Dmrta2 interacting with Zfp423 and the NuRD complex differentially repress the expression of Pax6 and Gsx1/2. Greater or lesser Dmrt repression capacity is indicated by thick or thin repression arrows, respectively. Similarly, Gsx1 and Gsx2 appear more sensitive to Pax6 than Wnt3a in the hem. Dmrt3 that is regulated by Dmrta2 contributes to the repression of Pax6 (De Clercq et al., 2016). Dmrta1 that is upregulated by Pax6 contributes to its ability to repress ventral telencephalic genes such as Ascl1 (Kikkawa et al., 2013). PSB, pallium subpallium boundary; dTel, dorsal telencephalon; vTel, ventral telencephalon.

Article Snippet: Then the membrane was blocked with Tris-buffered saline (TBS) with 5% non-fat milk for 1 h, at RT, and incubated at 4°C, O/N, with the following primary antibodies: rabbit anti-Flag (1:1,000); rabbit anti-HA (1:1,000); rabbit anti-H3 (1:50,000, Millipore, catalog #07-690); mouse anti-Myc (1:1,000, Sigma, catalog #M4439); mouse anti-Zfp423 (1:1,000); and the NurD complex antibody kit (Cell Signaling, catalog #8349T).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, Principle of the CANDDY technology. Each CANDDY molecule includes a target interactor (burgundy hexagon) and a CANDDY tag (yellow oval). The target protein is bound by the CANDDY molecule via the target interactor and is directly degraded by proteasome via the CANDDY tag. b, The CANDDY tag was synthesized by chemically inactivating the inhibitor site of MLM2238 (MLN). c, Structures of the RAS-SOS inhibitor (ref. 5), RAS-SOS-NH 2 , and TUS-007. d, KRAS G12D was mixed with DMSO, RAS-SOS inhibitor (4 µM) or TUS-007 (4 µM) and incubated under heating from 25 °C to 99 °C. The denature of KRAS G12D was monitored by the fluorescence. The typical curve of each group was shown. e, The correlation between relative amount of KRAS G12D and concentration of TUS-007 were approximated with Rodbard using the means of 3 independent examinations. The DC50 was estimated about 4 µM. f, TUS-007 induced KRAS G12D chemical knockdown by 26S proteasome in a cell free assay. KRAS G12D was incubated with 26S proteasome and agents as indicated for 3 h.

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Synthesized, Incubation, Fluorescence, Concentration Assay, Cell-Free Assay

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: The levels of chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) proteasome activities was monitored by Suc-LLVY-AMC, Bz-VGR-AMC, and Z-LLE-AMC, respectively. AMC fluorescence was monitored by a plate reader with excitation and emission filters of 360 and 460 nm, respectively (DMSO, 30 min = 1) (mean ± SEM; n = 2–3).

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: Successful KRAS G12V degradation by TUS-007. KRAS G12V protein was incubated with TUS-007 at the indicated concentrations in the presence of 26S proteasome for 1 h (mean ± SEM; n = 3). *P < 0.05 vs. DMSO.

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Incubation

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, TUS-007 induced chemical knockdown was independent of ubiquitination. GFP fused KRAS G12D and DsRed were transiently expressed in HeLa cells. The cells were treated with TUS-007 in the presence of a proteasome inhibitor (MLN), a ubiquitination inhibitor (NAEi) and a molecular chaperon inhibitor (HSP70 inhibitor: VER-155008) for 24 h. b & c, RAS-less MEFs expressing KRAS G12D, G12V or G12C were incubated with TUS-007 for 72 h. Representative blots of KRAS are shown (b). The relative amounts of KRAS mutants normalized to GAPDH are shown in the bar graph (mean ± SEM; n = 3–4). The cell viability was also measured (mean ± SEM; n = 3–4) (g). *P < 0.05 and ** P < 0.01 vs. DMSO. TUS-007 induced chemical knockdown did not cause non-specific cytotoxicity.

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Expressing, Incubation

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, KRAS G12D chemical knockdown induced by TUS-007 in SW1990 cells. SW1990 cells were treated with TUS-007 for 72 h and analyzed by immunoblotting. The KRAS G12D band intensity was normalized to that of GAPDH (mean ± SEM; n = 4). *P < 0.05 vs. DMSO. b, The proportion of annexin V-positive apoptotic SW1990 cells treated with DMSO, RAS-SOS-NH 2 or TUS-007 for 72 h (mean ± SEM; n = 3). *P < 0.05 vs. DMSO or RAS-SOS-NH 2 . c, TUS-007 increased the caspase 3/7 activity in SW1990 cells after treatment for 96 h (mean ± SEM; n = 3). *P < 0.05 and **P < 0.01 vs. DMSO. d, Caspase 3/7 activation by TUS-007 was proteasome dependent and ubiquitination independent. SW1990 cells were treated with the agents for 96 h (mean ± SEM; n = 3). **P < 0.01 vs. DMSO, ## P < 0.01 vs. TUS-007 (100 µM) only, and † no statistically significant difference compared with TUS-007 (100 µM) only. e, Tumor volume in mice with SW1990 cells transplanted subcutaneously and treated with p.o. administered TUS-007 or vehicle (mean ± SEM; n = 6–9). The agents were administered every 3 days. **P < 0.01 vs. vehicle alone. f, Body weight changes in mice with SW1990 cells transplanted subcutaneously (mean ± SEM; n = 6–9).

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Western Blot, Activity Assay, Activation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Highly expressed SERCA2 triggers tumor cell autophagy and is a druggable vulnerability in triple-negative breast cancer

doi: 10.1016/j.apsb.2022.05.009

Figure Lengend Snippet: SERCA2 tethers the ER with the isolation membrane through interaction with the ULK1/FIP200 complex and LC3B. (A) Co-IP of FLAG-SERCA2 with the ULK1/FIP200 complex members in transfected SUM1315 cells. (B) Co-localization of DFCP1 with SERCA2 in SUM1315 cells treated with or without EBSS for 1 h. Left panel, Representative images. Scale bar, 5 μm. Right panel, quantification of co-localization. n = 200 puncta. (C) Co-IP of LC3B with the ULK1/FIP200 complex members in SUM1315 cells transiently transfected with the indicated plasmids. (D) Western blot analysis of indicated proteins in SUM1315 cells transfected with the indicated siRNA or plasmids, which were then treated with EBSS for 12 h. (E) Co-localization of FIP200 with LC3 in SUM1315 cells transfected with the indicated siRNA or plasmids, which were then treated with EBSS for 1 h. Upper panel, representative images. Scale bars, 5 μm; insets, 2 μm. Lower panel, quantification of co-localization. n = 200 puncta. (F) Co-localization of DFCP1 with LC3 in SUM1315 cells transfected with the indicated plasmids, which were then treated with EBSS for 4 h in the absence or presence of 100 nmol/L thapsigargin (TG). Upper panel, representative images. Scale bars, 5 μm; insets, 2 μm. Lower panel, quantification of co-localization. n = 200 puncta. Data are shown as the mean ± SD of at least three independent experiments. ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Anti-LC3B (3868), anti-SERCA2 (9580), anti-WIPI2 (8567), anti-calnexin (2679) antibodies and Autophagy Induction (ULK1 Complex) Antibody Sampler Kit (46486) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Isolation, Co-Immunoprecipitation Assay, Transfection, Western Blot