Journal: Nature Communications

Article Title: RKIP regulates bone marrow macrophage differentiation to mediate osteoclastogenesis and H-type vessel formation

doi: 10.1038/s41467-025-62972-8

Figure Lengend Snippet: A Western blots of HIF-1α, HIF-2α and RKIP protein levels in BMMs from RKIP +/+ or RKIP -/- male mice under hypoxia culture for indicated time. B RKIP (green), HIF-1α (red) and DAPI (blue) immunofluorescence staining of BMMs from RKIP +/+ or RKIP -/- male mice under hypoxia culture for 24 h. C Western blots of HIF-1α and RKIP protein levels in BMMs following the adenovirus transduction of RKIP or the empty vector with the treatment of MG132 or chloroquine (CQ) under hypoxia culture for 48 h. D Western blots of the ubiquitination of HIF-1α in BMMs from RKIP +/+ or RKIP -/- male mice under hypoxia culture for 24 h, which were immunoprecipitated with anti-HIF1α. E BMMs were cultured under normoxia or hypoxia for 48 h; The interactions were detected through the indicated IP and IB analyses. F Western blots of HIF-1α, VHL, and RKIP in cytoplasmic and nuclear of BMMs from RKIP +/+ or RKIP -/- male mice under hypoxia culture for 48 h. G The illustration and images of proximity ligation assays (PLA) of indicated proteins in BMMs under hypoxia culture for 48 h. H Putative murine, rat, human, and canine RKIP NLS sequences, predicted from analysis with the PredictNLS program. I Flag(green), HIF-1α (red) and DAPI (blue) immunofluorescence staining of HEK-293T cells transfected with RKIP-Flag or RKIP-NLS-Del-Flag plasmid, which were under hypoxia culture for 24 h. J Western blots of HIF-1α, VHL, and Flag in cytoplasmic and nuclear of HEK-293T cells transfected with RKIP-Flag or RKIP-NLS-Del-Flag plasmid, which were under hypoxia culture for 48 h. K RKIP(green), HIF-1α (red) and DAPI (blue) immunofluorescence staining of BMMs transfected with NC or KPNB1 siRNA, which were under hypoxia culture for 24 h.

Article Snippet: The lysates were immunoprecipitated with Flag-Tag mouse mAb (#M1403-2, HUABIO, China, 1:100), HA-Tag mouse mAb (sc-53516, Santa Cruz, USA, 1:50), Myc-Tag mouse mAb (#30601ES60, YEASON, China, 1:100), RKIP rabbit mAb (#ET1610-18, HUABIO, China, 1:100), HIF-1α rabbit mAb (#36169, CST, USA, 1:100), mouse (G3A1) mAb IgG1 isotype control (#5415S, CST, USA) or rabbit (DA1E) mAb IgG isotype control (#3900S, CST, USA) according to experimental design at 4 °C overnight.

Techniques: Western Blot, Immunofluorescence, Staining, Transduction, Plasmid Preparation, Ubiquitin Proteomics, Immunoprecipitation, Cell Culture, Ligation, Transfection

Journal: Nature Communications

Article Title: RKIP regulates bone marrow macrophage differentiation to mediate osteoclastogenesis and H-type vessel formation

doi: 10.1038/s41467-025-62972-8

Figure Lengend Snippet: Schematic illustration showing the different roles of extranuclear or intranuclear RKIP in differentiation fate regulation of bone niche macrophages. RKIP is a critical molecule regulating the differentiation fate of bone niche macrophage via the RKIP/ARHGAP/CDC42 and RKIP/VHL/HIF-1α pathways.

Article Snippet: The lysates were immunoprecipitated with Flag-Tag mouse mAb (#M1403-2, HUABIO, China, 1:100), HA-Tag mouse mAb (sc-53516, Santa Cruz, USA, 1:50), Myc-Tag mouse mAb (#30601ES60, YEASON, China, 1:100), RKIP rabbit mAb (#ET1610-18, HUABIO, China, 1:100), HIF-1α rabbit mAb (#36169, CST, USA, 1:100), mouse (G3A1) mAb IgG1 isotype control (#5415S, CST, USA) or rabbit (DA1E) mAb IgG isotype control (#3900S, CST, USA) according to experimental design at 4 °C overnight.

Techniques:

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a Western blots of OMV-stimulated HeLa cells show specific autophagosome/lysosome-dependent degradation of mitochondrial marker proteins (TOM20 and TIM23), but not of Golgi (GM130) or endoplasmic reticulum (PDI). Western blots are representative of 3 independent experiments. b DiO-labeled gonococcal OMVs from strain ATCC 49226 colocalize with MitoBright-labeled mitochondria in HeLa cells. Scale bar, 5 μm. c Live HeLa cell microscopy and 3D image reconstruction shows OMVs remain associated with Cascade Blue-labeled endosomes when delivered to MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. d Gonococcal OMVs from strain ATCC 49226 dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P < 10 −15 . e TEM of HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86 show mitochondrial disruption and capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, one-way ANOVA with post-hoc Tukey test for mitochondrial disruption (Mock-ATCC 49226 OMVs: P = 2 × 10 −11 ; Mock-ZJXSH86 OMVs: P = 2 × 10 −11 ), Kruskal–Wallis with posthoc Dunn test for mitochondria in mitophagy-like structures (Mock-ATCC 49226 OMVs: P = 1 × 10 −6 ; Mock-ZJXSH86 OMVs: P = 7 × 10 −6 ). f Quantitative real-time PCR showing a reduced mitochondrial to genomic DNA ratio in ATCC 49226 OMV-stimulated HeLa cells. Data are mean ± s.d.; n = 3 independent biological replicates, one-way ANOVA with post-hoc Tukey test. g Increased HSP60 and LAMP1 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P = 6 × 10 −9 . h Increased HSP60 and LC3 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test (LC3 puncta Mock-OMVs: P = 7 × 10 −12 ; LC3 colocalized HSP60 Mock-ZJXSH86 OMVs: P = 4 × 10 −15 ). Cells in d , e , g , h are from 3 independent experiments. Images in b , c are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Western Blot, Marker, Labeling, Microscopy, Fluorescence, Membrane, Two Tailed Test, Disruption, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a TEM of HeLa cells expressing gonococcal PorB from strain ATCC 49226 show mitochondrial capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, two-tailed Mann–Whitney test, P = 2 × 10 −10 . b Western blots showing degradation of mitochondrial proteins TOM20 and TIM23 in HeLa cells expressing gonococcal PorB from strains ATCC 49226 and ZJXSH86, but not for PorB from Neisseria mucosa . c Western blots showing that gonococcal OMVs expressing PorB from N. mucosa lost the ability to induce degradation of TOM20 and TIM23 in HeLa cells. d Gonococcal OMVs expressing PorB from N. mucosa lost the ability to dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 55 cells from 4 independent experiments, Kruskal–Wallis with posthoc Dunn test (Mock-N.g. PorB: P = 8 × 10 −13 ; N.g. PorB-N.m. PorB: P < 10 −15 ). e Flow cytom e try analysis of TMRM fluorescence intensity in HeLa cells stimulated with gonococcal OMVs expressing gonococcal PorB or PorB from N. mucosa . f Gonococcal PorB structure (pdb entry 4AUI) and sequence of PorB from strain ATCC 49226. Lysines are indicated in blue, or in magenta when located within the PorB channel and associated with ATP (orange) binding. g Western blots showing reduced degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q. h Reduced HSP60 and LC3 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 2 × 10 −9 ; WT PorB-PorB K117Q: P = 1 × 10 −5 ). i Reduced HSP60 and LAMP1 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 9 × 10 −8 ). j Western blots of HeLa 4KO cells showing p62 expression restores PorB K117Q-dependent degradation of TOM20 and TIM23, while expression of OPTN or NDP52 restores PorB K5-dependent degradation of TOM20 and TIM23. k Western blots showing impaired degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q, K5 or K null . l Western blots showing that knock-down of PINK1 in HeLa cells inhibits PorB K5-dependent degradation of TOM20 and TIM23, but not for PorB K117Q. Cells in a , h , i are from 3 independent experiments. Western blots in b , c , g , j , k , l are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Fluorescence, Sequencing, Binding Assay, Knockdown

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a Western blots showing that knock-down of p62 in HeLa cells inhibits PorB K117Q-dependent degradation of TOM20 and TIM23. b Reduced p62 and HSP60 colocalization in HeLa cells expressing PorB K5 or K null compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; WT PorB-PorB K5: P < 10 −15 ; WT PorB-PorB K null : P < 10 −15 ). c Western blots of HeLa 4KO cells expressing WT or truncated p62 show that the p62 LIR domain and UBI domain are indispensable for PorB K117Q-induced degradation of TOM20 and TIM23. d Western blots showing co-immunoprecipitation of ubiquitin and p62 after immunoprecipitation of PorB WT and PorB K117Q. e Western blots showing co-immunoprecipitation of both K63-linked and K48-linked polyubiquitin, after immunoprecipitation of PorB from HeLa cells, which is enhanced with inhibitors for autolysosomal degradation (BafA1, 3-MA) for K63-linked ubiquitin or proteasomal degradation (MG132) for K48-linked ubiquitin. f Colocalization between HSP60 and K63-linked polyubiquitin, but not K48-linked polyubiquitin, is induced in HeLa cells expressing PorB. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test, K63 only Empty-PorB K117Q: P < 10 −15 . g Western blots after immunoprecipitation of HA-PorB from HeLa cells show co-immunoprecipitation of K63-linked polyubiquitin is dependent on PorB lysine 171 and co-immunoprecipitation of K48-linked polyubiquitin is dependent on PorB lysine 128. h Western blots showing PorB-induced degradation of TOM20 and TIM23 in HeLa cells is dependent on PorB lysines 117 and 171. i Western blots showing gonococcal OMVs expressing PorB K117Q/K171Q lost the ability to induce degradation of TOM20 and TIM23. Cells in b and f are from 3 independent experiments. Western blots in a , c , d , e , g , h , i are representative of 3 independent exper i ments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Western Blot, Knockdown, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a MS/MS spectrum of RNF213 peptide identified by mass spectrometry analysis of proteins co-immunoprecipitated with PorB from HeLa cells. b Western blots after immunoprecipitation of PorB from HeLa cells show co-immunoprecipitation of RNF213. c HeLa cells expressing PorB show colocalization between PorB, HSP60 and RNF213. Scale bar, 5 μm. d Live HeLa cell microscopy and 3D image reconstruction shows RNF213 surrounding PorB on MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. e Western blots showing PorB K171-dependent enhanced co-immunoprecipitation of ubiquitin from HeLa cells transfected with an RNF213 expression vector. f Western blots showing that transfection of HeLa cells with RNF213 siRNA abolishes PorB K171-dependent co-immunoprecipitation of ubiquitin. g Western blots showing PorB K117Q, but not PorB K171Q, enhances PorB-induced degradation of TOM20 and TIM23 in HeLa cells transfected with an RNF213 expression vector. h Western blots showing that transfection of HeLa cells with RNF213 siRNA prevents PorB-induced degradation of TOM20 and TIM23 for PorB K117Q, while degradation remains unaffected for PorB K171Q. Western blots in b, e – h are representative of 3 independent experiments. Images in c and d are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Tandem Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation, Western Blot, Expressing, Microscopy, Labeling, Fluorescence, Ubiquitin Proteomics, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: KLHL25-ACLY module functions as a switch in the fate determination of the differentiation of iTreg/Th17

doi: 10.1038/s42003-025-07917-z

Figure Lengend Snippet: a , b JNK inhibition reduces the transcription and protein level of Klhl25 . Naïve CD4 + T cells cultured under iTreg polarization condition were treated with specific inhibitors against Smad3, ERK, JNK, and p38 at the onset for 48 h. a Klhl25 mRNA level was assessed by qPCR. b KLHL25 protein level was analyzed by WB. c , d JNK inhibition attenuates the binding of CUL3-KLHL25 with ACLY and ACLY ubiquitination. Naïve CD4 + T cells transfected with pMIG- Klhl25 overexpression virus were cultured under iTreg polarization condition in the presence of JNK inhibitor (10 μM) for 48 h. Cells were treated with MG132 (10 μM) for 6 h before immunoprecipitated with antibodies against ACLY, and then the binding of ACLY with CUL3-KLHL25 was assessed by WB ( c ), the ubiquitination of ACLY was determined by WB ( d ). IgG serves as a negative control. e JNK inhibition suppresses TGFβ1-induced ACLY degradation. Naïve CD4 + T cells were transfected and cultured as described in the legend to ( c , d ) before the analysis of ACLY protein level by WB. ( f , g ) Impact of KLHL25 overexpression on CPT1 activity and iTreg differentiation upon JNK inhibition. Cells were transfected and cultured as described in the legend to ( c , d ) before analysis for CPT1 enzymatic activity assay by kit ( f ). Cells transfected and cultured as described in the legend to ( c , d ) for 72 h were stained for CD4 + Foxp3 + iTreg analyses by flow cytometry (FCM) (left) and quantified (right) ( g ). h Impact of Acly knockdown on iTreg differentiation upon JNK inhibition. Naïve CD4 + T cells transfected with Acly targeting siRNAs were cultured as described in the legend to ( g ) and then stained for CD4 + Foxp3 + iTreg analyses by FCM (left) and quantified (right). For ( a – h ), data are representative as mean ± SD (n = 3 biologically independent samples) with p values determined by one-way ANOVA test. For WB in ( b – h ), one representative experiment out of three is represented. The values indicate mean intensities based on three biological replicas.

Article Snippet: The antibodies for WB were anti-ACLY, anti-Ubiquitin, anti-CUL3, anti-NF-YA, anti-ERK, anti-JNK, anti-ERK-P (9101S, CST), anti-Smad3-P (9520S, CST), anti-JNK-P (4668, CST), anti-p38 (9212S, CST), anti-p38-p (9211S, CST), and anti-actin (HC201, TransGen).

Techniques: Inhibition, Cell Culture, Binding Assay, Ubiquitin Proteomics, Transfection, Over Expression, Virus, Immunoprecipitation, Negative Control, Activity Assay, Enzyme Activity Assay, Staining, Flow Cytometry, Knockdown

Journal: Virologica Sinica

Article Title: CK1α upregulates the IFNAR1 expression to prompt the anti-HBV effect of type I IFN in hepatoma carcinoma cells

doi: 10.1016/j.virs.2022.08.004

Figure Lengend Snippet: Effects of CK1α on the abundance of IFNAR1 and type I IFN signaling pathway. A Huh7 cells were pretreated by D4476 (5 ​μmol/L) or DMSO for 24 ​h and then stimulated by IFN-α (1000 IU/mL). The levels of IFNAR1, STAT1, STAT2 were analyzed by Western blot. B Huh7 cells were treated with 5 ​μmol/L D4476, then transfected with 1 ​μg HBV expression plasmids, followed by stimulation of IFN-α (1000 IU/mL). Cell lysates were analyzed by Western blot as indicated (left panel). The expression of IFNAR1 was detected by Western blot or qPCR (right panel). C The effect of overexpression of Flag-CK1α on the expression of IFNAR1 at transfection-replication model. Huh7 cells were co-transfected with 1 ​μg Flag-CK1α and 1 ​μg HBV expression plasmids, followed by stimulation of IFN-α (1000IU/mL) for 24 ​h. Cell lysates were analyzed by Western blot as indicated. D , E HepG2-NTCP cells were treated with 5 ​μmol/L D4476 or overexpression of Flag-CK1α, and infected with HBV particles (1000 GE/cell) and stimulated by IFN-α (1000 IU/mL). The levels of IFNAR1 were measured by Western blot. The relative level of IFNAR1/β-actin was calculated for the intensity measured by Image J software. Data represent the mean and standard deviation of three experiments. Statistical significance was determined by one-way ANOVA with Tukey post-hoc test (∗ P ​< ​0.05, ∗∗ P ​< ​0.01, ∗∗∗ P ​< ​0.001, NS, not significant).

Article Snippet: The following antibodies were used: CK1α (1:500, 55192-1-AP, rabbit, Proteintech, Rosemont, IL,USA), IFNAR1 (1:500, ab45172, rabbit, Abcam, Cambridge, MA, USA), STAT1 (1:1000, 14994, rabbit, CST, Danvers, MA, USA), pSTAT1 (1: 1000, 7649, rabbit, CST), STAT2 (1:1000, 72604, rabbit, CST), p-STAT2 (1:1000, 88410, rabbit, CST), Flag-tag (1:1000, MA-1-91878, mouse, Thermo, San Jose, CA, USA), HA-tag (1:1000, 26183, mouse, Thermo), β-actin (1:3000, sc-47778, mouse, Santa Cruz, Dallas, TX, USA), and ubiquitin (1:500, AF0306, rabbit, Beyotime, Shanghai, China).

Techniques: Western Blot, Transfection, Expressing, Over Expression, Infection, Software, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

doi: 10.3389/fphar.2017.00274

Figure Lengend Snippet: USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. (A) Comet assay was performed to assess DNA damage after 0.33 μM cisplatin treatment for 6 h in A549-WT, A549-USP22, and A549/CDDP cells (scale bar 20 μM). (B,C) H2A interacts with USP22 in A549-WT cells. A549-WT cells lysates were subjected to immunoprecipitation (IP) with control IgG, anti-USP22 (B) , and anti-H2A (C) antibodies. The immunoprecipitates were then blotted with the indicated antibodies. (D) H2A binding to USP22, γH2AX, and the ubiquitination of H2A (Lys119) was detected by IP and western blot after 0.33 μM cisplatin treatment for 48 h in A549-WT, A549/CDDP and A549-USP22 cells. (E) Western blot analysis of the proteins of USP22, γH2AX and Ubi-H2A after various concentrations of cisplatin treatment for 48 h. Every experiment was conducted at least three times, and the average is shown (mean ± SD). ∗ P < 0.05, ∗∗ P < 0.01, significant.

Article Snippet: Membranes were blocked in a buffer (TBS: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 5% bovine serum albumin and 0.1% Tween-20, followed by incubation with the primary antibodys USP22 (ab4812, 1:2000, Abcam, Cambridge, UK), Sirt1 (2496, 1:2000, CST, United States), H2AX (2595, 1:1000, CST, United States), γ-H2AX (Ser 139)(9718, 1:1000, CST, United States), Ubi-H2A (Lys119) (8240, 1:1000, CST, United States), Ku70 (10723-1-AP, 1:1000, Proteintech, United States), Bax (50599-2-lg, 1:1000, Proteintech, United States), cytochrome C (10993-1-AP, 1:1000, Proteintech, United States) or GAPDH (10494-1-AP, 1:3000, Proteintech, United States).

Techniques: Phospho-proteomics, Single Cell Gel Electrophoresis, Immunoprecipitation, Control, Binding Assay, Ubiquitin Proteomics, Western Blot