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: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: CIB1 forms a complex with EVER1 and EVER2. (A) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*) and EVER2 (D362* or T150Mfs*3), and comparison with those in heterozygous carriers, positive controls (ctrl 1 and 2), P4 and P11, and patients with RHOH and MST1 deficiencies. (B) CIB1, EVER1, and EVER2 mRNA levels were assessed by RT-qPCR in controls ( n = 4), CIB1 m/m (P1–P6 and P12), EVER1 m/m, and EVER2 m/m ( n = 3 each) LCLs. Each symbol represents the mean of one cell line measured in three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests relative to healthy controls. (C) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. Asterisk indicates a nonspecific band. (D) CIB1, EVER1, and EVER2 mRNA levels were measured by RT-qPCR in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), or CIB1 and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. The data were first normalized against RNaseP as a housekeeping gene and then against an appropriate untransduced parental cell line by the ΔΔCt method. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the corresponding untransduced control. (B and D) ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) HEK293T cells were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either separately or together; 24 h after transfection, samples were subjected to immunoprecipitation with FLAG (Fl)- or HA-specific antibodies (+). Samples incubated with nonspecific IgG served as specificity controls. Western blots were performed to detect coimmunoprecipitated CIB1-HA and FLAG-EVER1 or FLAG-EVER2. The immunoprecipitation of EVER1/2 and CIB1 was confirmed by reincubation with antibodies specific for FLAG and HA, respectively. The presence of all proteins was checked by Western blotting of an input sample taken before immunoprecipitation. GAPDH served as a loading control. vec, vector. (F) Healthy control keratinocytes were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either alone or in combination; 24 h after transfection, cells were subjected to immunofluorescence imaging with Alexa Fluor 568–HA and Alexa Fluor 488–FLAG antibody combinations. DAPI was used for counterstaining. Colocalization was assessed by calculating Pearson’s correlation coefficient with Imaris software. Bar, 13 µm. The results shown are representative of three independent experiments.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Derivative Assay, Comparison, Quantitative RT-PCR, Control, Retroviral, Transduction, Selection, Transfection, Immunoprecipitation, Incubation, Western Blot, Plasmid Preparation, Immunofluorescence, Imaging, Software

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Analysis of zinc signaling/levels and NF- k B activation in the presence and absence of CIB1. (A) HEK293T cells were transfected with plasmids encoding CIB1, EVER1, and EVER2 either alone or in combination and with a 4×MRE-dependent EGFP reporter construct. After 24 h, cells were stimulated overnight with PMA/ionomycin (10 ng/ml and 50 ng/ml, respectively) or zinc sulfate (ZnSO 4 ; 100 µM). The next day, cells were stained with 1 µg/ml DAPI to exclude dead cells, and GFP fluorescence was determined with an LSRII flow cytometer. The RRR with the value for vector-transfected cells was set at 100%. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the appropriate vector-transfected control (ns, P > 0.05; *, P < 0.05; ***, P < 0.001; n = 3). (B) Flow cytometric quantification of absolute amounts of labile zinc in LCLs derived from healthy controls, EVER1-, EVER2-, or CIB1-deficient patients, or in keratinocytes from P14 with 1 µM FluoZin-3 as described by . Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the healthy controls ( n = 3). (C) Kinetics of zinc flux in LCLs derived from healthy controls and EVER1-, EVER2-, or CIB1-deficient patients. Cells were loaded with 1 µM FluoZin-3 for 30 min. Fluorimetric measurement were performed on a Victor microplate reader. Baseline fluorescence was recorded every minute for 10 min. Cells were then loaded with 100 µM ZnSO 4 and recorded for 15 min. The specificity of the zinc signal was confirmed by adding the calcium-specific chelator BAPTA before the quenching of the signal with the zinc-specific chelator TPEN and recording for 20 min. No significant effect of genotype was detected in two-way repeat-measures ANOVA ( n = 3). (D) HEK293T cells were transfected with plasmids encoding CIB1-FLAG, FLAG-EVER1, and EVER2 either alone or in combination. Cells were stimulated with 50 ng/ml TNFα 6 h after transfection, incubated overnight, and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa and the noncanonical NF-κB component p100/p52. Expression of the constructs used for transfection was verified by incubation with a FLAG-specific antibody. GAPDH served as a loading control ( n = 3). (E) Primary keratinocytes from unrelated donors (controls 1 and 2), a healthy family member from kindred A1 carrying the mutation in a heterozygous state (A1.viii.2), and one patient each from kindreds A1 and C were stimulated with 10 ng/ml TNFα for 5, 10, or 20 min and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa. CIB1 levels were assessed with a polyclonal antibody. GAPDH served as a loading control. vec, empty vector ( n = 3).

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Activation Assay, Transfection, Construct, Staining, Fluorescence, Flow Cytometry, Plasmid Preparation, Comparison, Control, Derivative Assay, Incubation, Western Blot, Expressing, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: PLA and coimmunoprecipitation in HaCaT. (A) HaCaT cells were transfected with plasmids encoding FLAG-HPV5 E1, E2, E6, and E7, FLAG-HPV16 E1, E2, E5, E6, and E7, FLAG-HPV4 E8, FLAG–CRPV E8, and CIB1-HA alone or in combination. The day after transfection, samples were plated on microscopy slides, allowed to adhere, fixed in acetone, permeabilized, and subjected to Duolink PLAs with rabbit-HA– and mouse-FLAG–specific antibodies. Z stacks were acquired with a widefield microscope, and PLA-positive sites (defined as structures >0.35 µM 2 ) were scored with Imaris software for 15–50 cells per condition. These pooled results were obtained in two independent experiments. The orange bars indicate the mean. (B) HaCaT cells were transfected with plasmids encoding CIB1-HA and the FLAG-tagged HPV E ORFs scoring positive in the PLA in A. 1 d after transfection, samples were subjected to immunoprecipitation (IP) with FLAG-specific antibodies. Western blots were performed to detect coimmunoprecipitated HPV5 E1, HPV16 E2, E5, HPV4 E8, and CRPV E8. The immunoprecipitation of CIB1 was confirmed by reincubation with a FLAG-specific antibody. The presence of all proteins was checked by Western blotting analysis on an input sample taken before immunoprecipitation. GAPDH served as a loading control ( n = 3). vec, vector.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Transfection, Microscopy, Software, Immunoprecipitation, Western Blot, Control, Plasmid Preparation

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