Journal: Nature Communications

Article Title: A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences

doi: 10.1038/s41467-023-42746-w

Figure Lengend Snippet: Plasmids deposited at Addgene

Article Snippet: Primary antibodies used in this study: GFAP (1:1000, chicken, Rockland #200-901-D60); Aldh1l1 (1:400, rabbit, Abcam #Ab87117); Sox9 (1:1000, rabbit, Millipore Sigma #AB5535); NeuN (1:1000, chicken, Synaptic Systems #266 006); CD31 (1:100, rat, BD Biosciences #550274); V5 (1:400, human, Absolute Antibodies #AB00136-10.0); Myc (1:400, 488-labeled, Biotium #20436); FLAG (1:400, 543-labeled, Biotium #20433); HA (1:100, rat, Roche #11-867-423), and GFP (1:500, 488-labeled nanobody, ChromoTek #GBA488-100).

Techniques: Membrane, Plasmid Preparation, Expressing

Journal: Journal of cell science

Article Title: Improperly folded green fluorescent protein is secreted via a non-classical pathway.

doi: 10.1242/jcs.00047

Figure Lengend Snippet: Fig. 1. GFP is secreted via a non-classical pathway in CHO cells. (A) Untransfected CHO cells (Ctrl) and cells transiently transfected with either GFP (GFP) or preprolactin-myc (PPL) were labeled with [35S]-methionine and chased for 6 hours. The cell lysates (C) and media (M) were subjected to immunoprecipitation with either antibodies against GFP (lanes 1-8) or myc (lanes 9-12), and the washed immunoprecipitates were displayed by SDS-PAGE and fluorography. Some cells (+BFA) were treated with 1 µg/ml brefeldin A for 1 hour prior to labeling (lanes 3- 4, 7-8 and 11-12). ‘% Sec.’ denotes the percentage of secretion of GFP into the media as determined by a phosphorimager. (B) CHO cells transfected with plasmids encoding either mouse dihydrofolate reductase (mDHFR) or Schistosoma japonicum glutathione S-transferase (GST) both tagged with the FLAG epitope. The cells were labeled with [35S]-methionine and chased in serum-free medium. The cell lysate (C) and media (M) were immunoprecipitated with anti-FLAG antibodies and processed as above. (C) Plasmids coding for mDHFR and GFP were used to co-transfect CHO cells. The cells were labeled, chased and processed as described in B.

Article Snippet: Polyclonal antibodies against the FLAG epitope (DYKDDDDK) were purchased from ProSci.

Techniques: Transfection, Labeling, Immunoprecipitation, SDS Page, FLAG-tag

Journal: Vaccines

Article Title: The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System

doi: 10.3390/vaccines12040399

Figure Lengend Snippet: Displaying of the full-length SadA on the surface of S. typhimurium mutant cells. ( A ) Western blot of whole cell lysates to analyze expression of SadA in StmΔ sadA . The Flag tag was inserted at the N-terminus of SadA passenger. The recombinant protein bands were detected by incubating the PVDF using anti-Flag-tag antibody. The putative position of monomeric form (*) was indicated on the right side of the panel. Lane1, pTrc99A/StmΔ sadA ; lane 2, pSadA-Flag×3/StmΔ sadA . Dot blot of whole cells showed the surface display of Flag-tagged SadA on StmΔ sadA ( B ). Lane1, pTrc99A/StmΔ sadA ; lane2, pSadA-Flag×3/StmΔ sadA . ( C ) Cell surface display of Flag-tagged SadA by immunofluorescence staining using anti-Flag-tag primary antibody and AlexaFluor 488 conjugated secondary antibody (Objective, 100×; Magnification, 1000×).

Article Snippet: Subsequently, the membrane was incubated with anti-Flag tag rabbit polyclonal antibody (diluted to 1:3000 with 5% skim milk in PBST; Easybio, Beijing, China), or A1H10 or A3C10 (1:2000 dilution) for 1 h at 37 °C.

Techniques: Mutagenesis, Western Blot, Expressing, FLAG-tag, Recombinant, Dot Blot, Immunofluorescence, Staining

Journal: Vaccines

Article Title: The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System

doi: 10.3390/vaccines12040399

Figure Lengend Snippet: The full-length SadA fiber could display on the surface of S. typhimurium mutant cells with the assistance of SadB. ( A ) Western blot of whole cell lysates to analyze their expression in StmΔ sadA . The 1 mL induced cell suspensions (OD 600 = 1) were washed twice by PBS and resuspended in 200 μL SDS sample buffer and boiled for 5 min. Then, 60 μL pSadA-Flag×3/StmΔ sadA , 30 μL pSadBA-Flag×3/StmΔ sadA , and 30 μL pTrc99A/StmΔ sadA ran on 4~12% SDS-PAGE for Western blot. The recombinant protein bands were detected by incubating the PVDF with anti-Flag-tag antibodies. The putative position of monomeric form (*) was indicated on the right side of the panel. Lane1, pSadA-Flag×3/StmΔ sadA ; lane2, pSadBA-Flag×3/StmΔ sadA ; lane3, pTrc99A/StmΔ sadA . ( B ) Dot blot of whole cells to analyze the surface display of recombinant proteins on StmΔ sadA with the assistance of SadB or not. Lane1, pTrc99A/StmΔ sadA ; lane2, pSadA-Flag×3/StmΔ sadA ; lane3, pSadBA-Flag×3/StmΔ sadA . ( C ) Surface display capacity as revealed by immunofluorescence staining of the Flag-tag inserted N-terminus of SadA passenger in the presence of SadB (Objective, 100×; Magnification, 1000×). ( D ) A comparison of fluorescence intensities between the whole cells treated with protease K or not. (1) pSadA-Flag×3/StmΔ sadA ; (2) pSadBA-Flag×3/StmΔ sadA ; (3) pFM/StmΔ sadA ; (4) pTrc99A/StmΔ sadA . The data were presented as mean ± SD, and differences between groups were tested using one-way ANOVA. **** p < 0.0001, ns p > 0.05.

Article Snippet: Subsequently, the membrane was incubated with anti-Flag tag rabbit polyclonal antibody (diluted to 1:3000 with 5% skim milk in PBST; Easybio, Beijing, China), or A1H10 or A3C10 (1:2000 dilution) for 1 h at 37 °C.

Techniques: Mutagenesis, Western Blot, Expressing, SDS Page, Recombinant, FLAG-tag, Dot Blot, Immunofluorescence, Staining, Comparison, Fluorescence

Journal: Vaccines

Article Title: The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System

doi: 10.3390/vaccines12040399

Figure Lengend Snippet: Expressing and displaying epitopes on the cell surface using full-length and truncated SadAs. ( A ) Western blot showed that fused proteins could be expressed in the StmΔ sadA using the antibodies against Flag tag. Then, 30 μL pSadBA 1292 -FU2/StmΔ sadA (lane 1), pSadBA 877 -FU2/StmΔ sadA (lane 2), pSadBA 1171 -FU2/StmΔ sadA (lane 3), pSadBA 644 -FU2/StmΔ sadA (lane 4), pSadBA 269 -FU2/StmΔ sadA (lane 5) and pSadBA-FU2/StmΔ sadA (lane 6) treated with proteinase K (+) or not (−) were load on 4~12% SDS-PAGE for Western blot to show the changes of bands. The putative positions of monomeric (*) and trimeric (***) complexes were indicated under the bands. ( B ) Surface display capacity as revealed by immunofluorescence staining of the Flag-tag inserted N-terminus of SadA derivatives (Objective, 100×; Magnification, 1000×). ( C ) A comparison of fluorescence intensities between the whole cells treated with protease K or not. (1) pSadBA 1292 -FU2/StmΔ sadA ; (2) pSadBA 1171 -FU2/StmΔ sadA ; (3) pSadBA 877 -FU2/StmΔ sadA ; (4) pSadBA 644 -FU2/StmΔ sadA ; (5) pSadBA 269 -FU2/StmΔ sadA ; (6) pSadBA-FU2/StmΔ sadA ; (7) pTrc99A/StmΔ sadA . The data were presented as mean ± SD, and differences between groups were tested using one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05.

Article Snippet: Subsequently, the membrane was incubated with anti-Flag tag rabbit polyclonal antibody (diluted to 1:3000 with 5% skim milk in PBST; Easybio, Beijing, China), or A1H10 or A3C10 (1:2000 dilution) for 1 h at 37 °C.

Techniques: Expressing, Western Blot, FLAG-tag, SDS Page, Immunofluorescence, Staining, Comparison, Fluorescence

Journal: Vaccines

Article Title: The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System

doi: 10.3390/vaccines12040399

Figure Lengend Snippet: Recombinant proteins displaying on the surface of cells using truncated SadA as an anchoring motif. Western blot showed that fused proteins could be expressed in the StmΔ sadA using the antibodies against Flag tag ( A ). The putative positions of monomeric (*), dimeric (**), and trimeric (***) complexes were indicated under the bands. (1) pSadBA 1292 -FM/StmΔ sadA ; (2) pSadBA 1292 -FUM/StmΔ sadA ; (3) pSadBA 1292 -FUPM/StmΔ sadA ; (4) pSadBA 877 -FM/StmΔ sadA ; (5) pSadBA 877 -FUM/StmΔ sadA ; (6) pSadBA 877 -FUPM/StmΔ sadA ; (7) pFM/StmΔ sadA . ( B ) Cell surface display of Flag-tagged SadA derivatives by immunofluorescence staining using anti-Flag-tag primary antibody and Alexa Fluor 488-conjugated secondary antibody (Objective, 100×; Magnification, 1000×). ( C ) A comparison of fluorescence intensities between the whole cells treated with protease K or not. (1) pSadBA 1292 -FU2/StmΔ sadA ; (2) pSadBA 1292 -FM/StmΔ sadA ; (3) pSadBA 1292 -FUM/StmΔ sadA ; (4) pSadBA 1292 -FUPM/StmΔ sadA ; (5) pSadBA 877 -FU2/StmΔ sadA ; (6) pSadBA 877 -FM/StmΔ sadA ; (7) pSadBA 1292 -FUM/StmΔ sadA ; (8) pTrc99A/StmΔ sadA . The data were presented as mean ± SD, and differences between groups were tested using one-way ANOVA. * p < 0.05, **** p < 0.0001, ns p > 0.05.

Article Snippet: Subsequently, the membrane was incubated with anti-Flag tag rabbit polyclonal antibody (diluted to 1:3000 with 5% skim milk in PBST; Easybio, Beijing, China), or A1H10 or A3C10 (1:2000 dilution) for 1 h at 37 °C.

Techniques: Recombinant, Western Blot, FLAG-tag, Immunofluorescence, Staining, Comparison, Fluorescence