Journal: bioRxiv

Article Title: RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis

doi: 10.1101/2024.09.16.613344

Figure Lengend Snippet: (A) HUVECs depleted of RalA or RalB using specific siRNAs and compared with control siRNA. Western blot of total cell lysates showing RalA and RalB knockdown after 48 hours of transfection of siRNA, with β -actin as loading control. (B) Bar graphs showing total vWF antigen in media as estimated by ELISA 30 minutes after 1 U/ml thrombin stimulation 48 hours after siRNA transfection (** P <0.01; from 3 individual experiments each with duplicates). (C) HUVECs treated with increasing concentrations of dihydroartemisinin for 2 hours or DMSO. Western blot showing degradation of RalB but not RalA by dihydroartemisinin, with β-actin as loading control. (D) Bar graphs showing total vWF antigen in media as estimated by ELISA 30 minutes after 1 U/ml thrombin stimulation 2 hours after dihydroartemisinin treatment (** P <0.01, *** P <0.001; **** P <0.0001 from 3 individual experiments each with duplicates). (E) Total cell lysates of resting and thrombin-treated (1 U/ml for 2 minutes) HUVECs were incubated with RalBP1-RBD conjugated agarose slurry and eluates immunoblotted with anti-RalB antibody to determine total GTP-loaded RalB in each condition. Coomassie stain showing RalBP1-RBD as loading control (left). Bar graphs showing densitometric analysis (right). (** P <0.01; from 3 individual experiments).

Article Snippet: Experiments were performed on RalB and RalA knockdown cells 48 h post-transfection. pLA-CMV-N-Flag-RalBWT (plasmid#50990), pLX301 (plasmid#25895), pEGFP-C2-CD63 (plasmid#62964) were obtained from Addgene.

Techniques: Control, Western Blot, Knockdown, Transfection, Enzyme-linked Immunosorbent Assay, Incubation, Staining

Journal: Journal of Microbiology and Biotechnology

Article Title: Engineering of Recombinant Human Papillomavirus 16 L1 Protein for Incorporation with para -Azido- L -Phenylalanine

doi: 10.4014/jmb.2407.07033

Figure Lengend Snippet: Details of the plasmids, primers, and strains used in this study.

Article Snippet: pEVOL-pAzF , pEVOL plasmid harboring two copies of synthetase and one copy of tRNA for para -azido- L -phenylalanine , Addgene (ID: 31186).

Techniques: Plasmid Preparation, Sequencing, Cloning, Expressing, Mutagenesis

Journal: Journal of Microbiology and Biotechnology

Article Title: Engineering of Recombinant Human Papillomavirus 16 L1 Protein for Incorporation with para -Azido- L -Phenylalanine

doi: 10.4014/jmb.2407.07033

Figure Lengend Snippet: Black arrows indicate bands corresponding to the truncated form of each MBP-HPV16L1 Amb candidate, while white arrows outlined in black indicate bands corresponding to pAzF-containing MBP-HPV16L1 proteins. M, molecular marker; WT, E. coli BL21 (DE3); L1, E. coli BL21 (DE3) pMALc2XHPV16L1; -pAzF, each induced candidate without pAzF; +pAzF, each induced candidate with pAzF. E. coli BL21 (DE3) (pMALc2X-HPV16L1/Amb1, pEVOL-pAzF) ( A ) E. coli BL21 (DE3) (pMALc2X-HPV16L1/Amb2, pEVOL-pAzF) ( B ) E. coli BL21 (DE3) (pMALc2X-HPV16L1/Amb3, pEVOL-pAzF) ( C ) E. coli BL21 (DE3) (pMALc2X-HPV16L1/Amb4, pEVOLpAzF) ( D ) and E. coli BL21 (DE3) (pMALc2X-HPV16L1/Amb5, pEVOL-pAzF) ( E ) respectively. ( F ) Fluorescence image from MBP-HPV16L1 F505γ conjugated with DBCO-PEG 3 -FITC.

Article Snippet: pEVOL-pAzF , pEVOL plasmid harboring two copies of synthetase and one copy of tRNA for para -azido- L -phenylalanine , Addgene (ID: 31186).

Techniques: Marker, Fluorescence

Journal: Nature cell biology

Article Title: TBKBP1 and TBK1 form a growth factor signaling axis mediating immunosuppression and tumorigenesis

doi: 10.1038/s41556-019-0429-8

Figure Lengend Snippet: Growth factors activate TBK1 in a TBKBP1-dependent manner. a,b , Immunoblot analysis of S172-phosphorylated (p-) and total TBK1 in the lysates of HEK293T cells transfected with TBK1 along with Kras G12D ( a ) or RalB ( b ). c,d , Immunoblot analysis of TBK1 S172 phosphorylation in EGF-induced A549 cells stably transduced with vector control or Kras G12D ( c ) and RalB ( d ). e , Immunoblot analysis of S172-phosphorylated (p-) and total TBK1 in whole-cell lysates of A549 cells stimulated by EGF, Insulin or FBS. f,g, Immunoblot analysis of S172-phosphorylated (p-) and total TBK1 in whole-cell lysates of EGF-stimulated lung cells from WT or Tank KO mice ( f ) or EGF-stimulated A549 cells stably transduced with a control shRNA (shCtrl) or shRNAs for Tank ( g , upper panel) and Nap1 ( g , lower panel). h,i , Immunoblot analysis of S172-phosphorylated TBK1 (p-TBK1) and the indicated other proteins in whole-cell lysates of control or Tbkbp1 -knockdown A549 cells stimulated with the indicated growth factors ( h ) or PRR ligands ( i ). Data are representative of three independent experiments. Unprocessed blots are shown in .

Article Snippet: Flag-tagged TBK1 were provided by Dr. Chen Wang (Shanghai Institutes for Biological Sciences). pBabe-HA-Kras-G12D, pLA CMV-HA RALB, pRK5-HA-Raptor and pRK-5-Myc-Rictor were purchased from Addgene.

Techniques: Western Blot, Transfection, Phospho-proteomics, Stable Transfection, Transduction, Plasmid Preparation, Control, shRNA, Knockdown