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: Redox Biology

Article Title: Mechano-activated connexin hemichannels and glutathione transport protect lens fiber cells against oxidative insults

doi: 10.1016/j.redox.2024.103216

Figure Lengend Snippet: Homomeric and heteromeric HCs formed by Cx50 and Cx46 are induced open by FFSS and inhibited by Cx50 dominant-negative mutants. CEF cells were infected with high-titer RCAS(A) vehicle (V) or recombinant RCAS(A) containing Cx50, Cx50P88S, Cx50E48K, Cx50H156N, Cx46 or co-infected with RCAS(A) containing Cx46 and Cx50 or Cx50H156N. (A) Membrane extracts were immunoblotted with anti-Flag for detecting exogenous Cxs or β-actin as a loading control. (B) CEF cells were subjected to FFSS at 1 dyn/cm 2 or static condition for 30 min and then incubated with 0.4 % LY/RD for 10 min. The images of dye uptake are shown (upper panel) and the LY uptake percentage was quantified by subtracting LY/RD double-positive cells of LY positive cells (lower panel). (n = 3). Scale bar: 100 μm * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Two-way ANOVA).

Article Snippet: Rabbit anti-Flag monoclonal antibody (#600-401-383) was obtained from Rockland (Limerick, PA, USA).

Techniques: Dominant Negative Mutation, Infection, Recombinant, Membrane, Incubation

Journal: Redox Biology

Article Title: Mechano-activated connexin hemichannels and glutathione transport protect lens fiber cells against oxidative insults

doi: 10.1016/j.redox.2024.103216

Figure Lengend Snippet: Cx50 HCs activated by FFSS have a higher capacity for transporting GSH. (A) CEF cells were infected with high-titer recombinant RCAS(A) retrovirus containing Cx50, Cx46 or co-infected with RCAS(A) containing Cx46 and Cx50. Membrane extracts were immunoblotted with anti-Flag or β-actin antibody (left panel). The relative ratio of band intensity of Cx50/Cx46/Cx46 + Cx50 to β-actin was quantified, with the Cx50 to β-actin ratio normalized to 1 (right panel). (B) CEF cells were subjected to FFSS at 1 dyn/cm 2 or static condition for 30 min, treated with 1 mM GSH for different time periods, and then incubated with 10 μM ThiolTracker™ for 30 min (n = 3). GSH uptake after FFSS was quantified by subtracting the fluorescence intensity of non-FFSS groups. (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to non-GSH treated control (One-way ANOVA). # P < 0.05, ## P < 0.01, Cx46 or Cx46 + Cx50 compared to Cx50 at the same time point (One-way ANOVA). (C) CEF cells were subjected to FFSS at 1 dyn/cm 2 for 30 min, treated with various concentrations of GSH for 20 min, and then incubated with 10 μM Thioltracker™ for 30 min (n = 3). Data were analyzed by the Michaelis-Menten equation and fitted by non-linear regression analyses. Vmax (AU/20 min, upper panel) and Km (mM, lower panel) are presented. ** P < 0.01, *** P < 0.001, comparison of Vmax between Cx50 and Cx46 or Cx46 + Cx50 (One-way ANOVA). For (B) and (C) , GSH uptake level was normalized by the relative ratio of protein expression of Cx50/Cx46/Cx46 + Cx50 to β-actin. (D) CEF cells infected with RCAS(A) vehicle (V) or recombinant RCAS(A) containing Cx50, Cx50P88S, Cx50E48K, Cx50H156N, Cx46 or co-infected with RCAS(A) containing Cx46 and Cx50 or Cx50H156N were treated with or without FFSS at 1 dyn/cm 2 for 30 min, treated with 1 mM GSH for 20 min, and then incubated with 10 μM Thioltracker™ for 30 min (n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Two-way ANOVA). (E) Differentiated lens primary cells were infected with RCAS(A) (V) or recombinant RCAS(A) containing Cx50 or Cx50H156N. Membrane extracts were immunoblotted with anti-Flag, anti-Cx50, or β-actin antibodies. (F) Differentiated lens primary cells infected with recombinant RCAS(A) containing Cx50 or Cx50H156N were subjected to FFSS at 1 dyn/cm 2 or static condition for 30 min, treated with 1 mM GSH for 30 min, and then incubated with 10 μM Thioltracker™ for 30 min (n = 3). Scale bar: 100 μm ** P < 0.01, **** P < 0.0001 (Two-way ANOVA).

Article Snippet: Rabbit anti-Flag monoclonal antibody (#600-401-383) was obtained from Rockland (Limerick, PA, USA).

Techniques: Infection, Recombinant, Membrane, Incubation, Fluorescence, Comparison, Expressing

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

Journal: Scientific Reports

Article Title: G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

doi: 10.1038/srep43480

Figure Lengend Snippet: ( a ) Western blot and immunoprecipitation (IP) analysis of FLAG-HA-Emerald GFP (FHG) tagged with C-terminal G196-tag in HeLa cells using mAb G196. HeLa cells were transfected with FHG/pcDNA3 or FHG-G196/pcDNA3. After incubation for two days, the cells were lysed and subjected to Western blotting with anti-FLAG (M2) or G196 mAbs ( left panel ). These lysates were immunoprecipitated with anti-FLAG (M2) (lanes 7, 8) or G196 (lanes 9, 10) mAbs and subjected to Western blotting with anti-FLAG polyclonal antibody ( right panel ). ( b ) Immunofluorescence of GFP- and G196-tagged nuclear protein in HeLa cells using mAb G196. HeLa cells were transfected with FHG-G196-NAC1/pcDNA3. After incubation for two days, the cells were fixed and incubated with mAb G196 and anti-GFP polyclonal Ab, then stained with Alexa-594 anti-mouse and Alexa-488 anti-rabbit secondary antibodies. ( c ) Western blot and chromatin immunoprecipitation (ChIP) analysis of Atf1-G196-GFP in fission yeast using mAb G196. A DNA fragment containing the transcription factor atf1 gene fused with the G196 epitope- GFP gene was replaced with the genomic atf1 gene. Protein extracts were separated by SDS-PAGE and detected with mAb G196 and anti-GFP polyclonal antibody ( left panel ). GFP-Iws1 expressing cells were used as a negative control. Asterisk indicates non-specific band also detected using the second Ab alone. Schematic representation of the tdh1 locus ( right upper panel ). The promoter of the tdh1 gene harbors a CRE consensus site. ChIP-qPCR analysis of the Arf1-G196-GFP level in the indicated regions ( right lower panel ). ( d ) Immunofluorescence of GFP- and G196-tagged nuclear protein in fission yeast cells using mAb G196. Atf1-G196-GFP expressing cells were fixed and incubated with mAb G196 and anti-GFP polyclonal Ab, then stained with Alexa-594 anti-mouse and Alexa-488 anti-rabbit secondary antibodies.

Article Snippet: The following commercial antibodies were used: mouse monoclonal anti-FLAG (M2, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal anti-FLAG (60–031, BioAcademia, Osaka, Japan) and anti-GFP (60–011, BioAcademia); HRP-conjugated goat F(ab’) 2 anti-mouse (710–1332, Rockland Immunochemicals, Limerick, ME, USA) and goat anti-rabbit IgG (111–035–003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA); Alexa 488-conjugated goat anti-rabbit IgG(H+L) (A-11034, ThermoFisher Scientific, Waltham, MA, USA) and Alexa 594-conjugated goat anti-mouse IgG(H+L) (A-11032, ThermoFisher Scientific).

Techniques: Western Blot, Immunoprecipitation, Transfection, Incubation, Immunofluorescence, Staining, Chromatin Immunoprecipitation, SDS Page, Expressing, Negative Control, ChIP-qPCR