Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunoprecipitation showing the interaction of FBXO22 with SDCBP in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( B ) Immunoprecipitation showing the interaction of SDCBP with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( C ) Co-immunoprecipitation showing the interaction of FBXO22 with BACH1 in HEK293 cells with or without SDCBP after the indicated transfections. ( D ) Schematic of experimental design to investigate the assembly of SCF FBXO22 –BACH1 complex via His Pull-down assay and endogenous IP assay in Fig. D– . ( E ) His-pulldown assay showing the interaction of FBXO22 with SKP1 in HEK293 cells with control vector or Myc-SDCBP-expressing vector after the indicated transfections. See also Appendix Fig. S . ( F ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in scramble and in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1, PTEN, and PD-L1 protein expression in A549 cells transfected with scramble or SDCBP siRNA. ( H ) Western blot showing BACH1 and PD-L1 protein expression in NCI-H1299 cells transfected with scramble or SDCBP siRNA. ( I ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( J ) In vivo ubiquitylation assay showing the inhibitory effect of SDCBP on SCF FBXO22 -mediated K48-linked polyubiquitylation of BACH1 in HEK293 cells transfected with the indicated plasmids.

Article Snippet: pCMV3-C-His-SKP1 cDNA , Sino Biological , Cat#HG14161-CH.

Techniques: Immunoprecipitation, Transfection, Pull Down Assay, Control, Plasmid Preparation, Expressing, Western Blot, In Vivo, Ubiquitin Assay

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Crosslink immunoprecipitation showing an interaction of FBXO22 with SDCBP in Hs578T cells transfected with Myc-SDCBP-expressing vector or Myc-SDCBP_Δ4-expressing vector. Schematic showing the FBXO22 mutant constructs generated using the HA-FBXO22 plasmid. ( C ) Immunoprecipitation showing SDCBP interactions with FBXO22 and its mutant constructs in HEK293 cells transfected with the indicated plasmids. ns indicates non-specific bands. See also Fig. , . ( D ) His-Pulldown assay showing the effect of SDCBP on SKP1-CUL1-FBXO22 complex formation after the indicated transfections in HEK293 cells. See also Appendix Fig. S . ( E ) His-Pulldown assay showing the effect of SDCBP KO on the SCF FBXO22 –BACH1 complex formation in the scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or His-SKP1-expressing vector. See also Appendix Fig. S . ( F ) Immunoprecipitation showing the effect of SDCBP KD on the SCF FBXO22 –BACH1 complex formation in MDA-MB-231 cells transfected with scramble or SDCBP siRNA. ( G ) Immunoprecipitation showing the effect of SDCBP overexpression on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with control vector or Myc-SDCBP-overexpressing vector. ( H ) Immunoprecipitation showing the effect of SDCBP PDZ1 domain on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with a control vector or a Myc-SDCBP-PDZ1-overexpressing vector. .

Article Snippet: pCMV3-C-His-SKP1 cDNA , Sino Biological , Cat#HG14161-CH.

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Generated, Control, Over Expression

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: Schematic showing the novel oncogenic roles of SDCBP in promoting aggressiveness and mitochondrial inhibitor resistance in TNBCs. An abundance of SDCBP stabilizes the BACH1 protein by blocking E3 ubiquitin ligase SCF FBXO22 complex-targeted BACH1 for degradative ubiquitination. Mechanistically, SDCBP binds to different members of the SCF FBXO22 complex, including SKP1 and FBXO22, via its PDZ1 domain and induces SCF FBXO22 complex disassociation, suggesting that SDCBP is a key adapter regulating the activity of the E3 ubiquitin ligase SCF FBXO22 complex in the proteasomal pathway. SDCBP-induced BACH1 accumulation upregulates several pro-metastatic genes and downregulates numerous mitochondrial ETC genes, resulting in tumor progression and high resistance to metformin treatment in TNBCs. Targeting SDCBP switches the SCF FBXO22 complex to degrade BACH1 protein via the proteasome, reducing tumor aggressiveness and boosting the anti-tumor effect of metformin administration in TNBCs. .

Article Snippet: pCMV3-C-His-SKP1 cDNA , Sino Biological , Cat#HG14161-CH.

Techniques: Blocking Assay, Ubiquitin Proteomics, Activity Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation.

doi: 10.1096/fj.201801681R

Figure Lengend Snippet: Figure 2. NLRP3 interacts with CUL1 independent of ROC1 and Skp1. A) The lysates of PMA-differentiated THP-1 macro- phages were immunoprecipitated (IP) with anti-NLRP3 antibody and then immunoblotted (IB) with indicated antibodies. B) HEK293T cells were transfected with pCAGGS-HA-NLRP3 along with pcDNA3.1(+)-3xFlag-Cul1, pcDNA3.1(+)-3xFlag-ROC1, pcDNA3.1(+)-3xFlag-SKP1-a, and pcDNA3.1(+)-3xFlag-SKP1-b. C) HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pcDNA3.1(+)-3XFlag-Cul1. D) HEK293T cells were transfected with pcDNA3.1(+)-3xFlag-Cul1 and pCAGGS-HA-NLRP3, pcDNA3.1(+)- 3xFlag-ROC1 and pcDNA3.1(+)-3xFlag-SKP1, pcDNA3.1(+)-3XFlag-SKP1 and pCAGGS-HA-NLRP3, or pcDNA3.1(+)-3xFlag-ROC1 and pCAGGS-HA-NLRP3. E) HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cul1 or pcDNA3.1(+)-3XFlag-ROC1 and pCAGGS-HA-NLRP3 at different concentrations. F) HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1 (+)-3xFlag-Cul1 and pcDNA3.1(+)-3xFlag-ROC1 at different concentrations. G) HEK293T cells were transfected with pcDNA3.1- Flag-Cullin1 and pcDNA3.1-NLRP3 along with pSilencer2.1-U6-shNC or pSilencer2.1-U6-shROC1#2. H) HEK293T cells were transfected with pcDNA3.1-3XFlag-Cullin1 and pcDNA3.1-NLRP3 along with pSilencer2.1-U6-shNC, pSilencer2.1-U6-shCUL1#1, pSilencer2.1-U6-shROC1#2, or pSilencer2.1-U6-shSKP1#1. B–H) The cell lysates were immunoprecipitated with anti-Flag antibody (B, C, G, H), anti-NLRP3 antibody (C), and anti-Cul1 antibody (D–F) and immunoblotted with indicated antibodies.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Transfection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation.

doi: 10.1096/fj.201801681R

Figure Lengend Snippet: Figure 3. CUL1 represses NLRP3 inflammasome in HEK293T cells. A) HEK293T cells were transfected or cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC. B) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-procaspase-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3Flag-ATP1b3, pcDNA3.1(+)-3Flag-PGM1, or pcDNA3.1(+)-3Flag-Cul1. Secreted IL-1b in the supernatants were analyzed by ELISA. C) Diagrams of CUL1 and CUL1DC (upper). HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cul1 or pcDNA3.1(+)-3XFlag- Cul1DC, as indicated. Secreted IL-1b in the supernatants were analyzed by ELISA. D) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag- Cul1 or pcDNA3.1(+)-3XFlag-Cul1DC. E) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-ASC, or pcDNA3.1(+)-pro–Casp-1 along with pcDNA3.1(+)-3XFlag-Cul1 for 24 h. The cells were treated with nigericin (10 mM) for 2 h. F) HEK293T cells were cotransfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag-Cul1, pcDNA3.1(+)-3XFlag- Cul2, or pcDNA3.1(+)-3XFlag-Cul3. The cell lysates were immunoprecipitated (IP) with anti-Flag antibody and immunoblotted (IB) with indicated antibodies. G) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)-pro– Casp-1, and pcDNA3.1(+)-pro-IL-1b along with pcDNA3.1(+)-3XFlag-Cul1, pcDNA3.1(+)-3XFlag-Cul2, or pcDNA3.1(+)-3XFlag-Cul3. H) HEK293T cells were transduced with lentiviruses stably expressing shRNA (sh-NC) and shRNA against Cul1 (sh-Cul1#1 and sh- Cul1#2) and selected with puromycin for 2 wk. CUL1 and GAPDH mRNAs were determined by quantitative RT-PCR (upper), and Cul1 and GAPDH proteins were detected Western blot analyses (lower). I) HEK293T cells stably expressing sh-NC or sh-Cullin1#2 were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cul1. J) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)- pro-IL-1b, or pcDNA3.1(+)-pro–Caspase-1 along with pcDNA3.1(+)-3XFlag-Cullin1, pcDNA3.1(+)-3XFlag-ROC1, or pcDNA3.1(+)- 3XFlag-Skp1. Secreted IL-1b in the supernatants were analyzed by ELISA. A, D, E, G, I, J) Secreted IL-1b in the supernatants were analyzed by ELISA (upper). Matured IL-1b (p17) and matured Casp-1 (p20) in the cell supernatants were determined by immunoblot analyses with indicated antibodies (lower). A, D, I) Data shown are means 6 SEM. N.s., not significant. *P , 0.05, **P , 0.01, ***P , 0.0001.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Transduction, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: Elevated FBXO45 promotes liver tumorigenesis through enhancing IGF2BP1 ubiquitination and subsequent PLK1 upregulation

doi: 10.7554/eLife.70715

Figure Lengend Snippet:

Article Snippet: antibody , anti-SKP1 (Rabbit Polyclonal) , Proteintech , Cat. #: 10990–2-AP;RRID: AB_2187492 , WB (1:1000).

Techniques: Knock-In, Isolation, Control, Recombinant, Immunoprecipitation, Mutagenesis, Software

Journal: STAR Protocols

Article Title: Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing

doi: 10.1016/j.xpro.2023.102340

Figure Lengend Snippet: Preparation of GST-eIF4E K119A , input RNA and cap-purified RNA (A) Small fraction of samples in the indicated steps are resolved by SDS-PAGE and proteins were visualized with CBB. IPTG(-): before induction, IPTG(+): after induction, Sonicated: sonicated sample before centrifugation, Cleared sup: sonicated sample after centrifugation, Flow-through: proteins unbound to glutathione 4B Sepharose, Eluted1/2/3: proteins eluted using glutathione, Beads: proteins remained on the beads after three consecutive elution. (B) EtBr staining showing efficient removal of small RNAs by two consecutive LiCl precipitation. ppt: precipitated, sup: unprecipitated fraction was ethanol-precipitated and then analyzed. (C) CBB staining of samples after glutathione/3C protease elution of capped RNAs. (D) RNA samples in the indicated steps are resolved by agarose gel electrophoresis and stained with SYBR Gold. Recombinant GST protein was used in the control pull down experiments.

Article Snippet: pGEX6P-1/eIF4E K119A , Addgene , Addgene: 199440.

Techniques: Purification, SDS Page, Sonication, Centrifugation, Staining, Agarose Gel Electrophoresis, Recombinant, Control

Journal: STAR Protocols

Article Title: Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing

doi: 10.1016/j.xpro.2023.102340

Figure Lengend Snippet:

Article Snippet: pGEX6P-1/eIF4E K119A , Addgene , Addgene: 199440.

Techniques: Virus, Recombinant, Protease Inhibitor, RNA Sequencing, Software