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rabbit anti flag pab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti flag pab
    a Immuno-TLC of extracted gangliosides from CHO-Δ Slc33a1 (Ex1-5 del) cells transfected with a plasmid encoding human V5-tagged ST8SIA1, alone or in combination with a plasmid encoding the indicated N-terminally Flag-tagged SLC33A1 variants. Mock transfected cells were used as negative control. Total gangliosides were extracted and separated by TLC followed by staining with anti-9- O -Ac-GD3 mAb M-T6004 or anti-GD3 mAb R24. Representative plates from one of three independent experiments are shown. b Flow cytometric analysis of CHO-Δ Slc33a1 (Ex1-5 del) cells transiently transfected as described in ( a ) and stained with anti-9- O -Ac-GD3 mAb M-T6004. Positive cells were gated as shown in Supplementary Fig. . Data are presented as mean ± s.d. of n = 4 independent experiments. One-way ANOVA followed by Bonferroni’s post-hoc test. **** p < 0.0001 indicates a significant difference in comparison to ST8SIA1/SLC33A1-WT expressing cells. Western blot analysis was performed to validate the expression of all Flag-tagged SLC33A1 variants. Protein bands representing full-length SLC33A1 and the C-terminally truncated variant p.Y366* are marked by an arrow. The bands marked by asterisks are already present in the lysate of mock- and ST8SIA1 -transfected cells and represent non-specific cross-reactivity of <t>the</t> <t>anti-Flag</t> antibody with endogenous cellular proteins. Actin was used as loading controls. c Cartoon representation of the cryo-EM structure of SLC33A1 (PDB ID 9M0S) . The N- and C-terminal six-helix bundles of SLC33A1 are colored in green and beige, respectively. Side chains of the residues in position 110 and 113 are shown as sticks. Dotted red lines visualize the course of the helix axes of TMH2, TMH11, and TMH12. Detail enlargements of the SLC33A1 structure highlighting the positioning of A110 ( d ) and G509 ( e ). Helices are shown as semi-transparent ribbons with selected residues shown in stick representation. Hydrogen bonds are indicated as dotted lines. For clarity, only the main chain atoms of L474 and V471 are depicted. f Schematic representation of the membrane topology of SLC33A1. Positions of patient-derived SLC33A1 mutations are indicated. Source data and exact p values are provided as a file.
    Rabbit Anti Flag Pab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+anti+flag+pab/pmc13043746-439-11-21
    Average 86 stars, based on 1 article reviews
    rabbit anti flag pab - by Bioz Stars, 2026-09
    86/100 stars

    Images

    1) Product Images from "Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis"

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis

    Journal: Nature Communications

    doi: 10.1038/s41467-026-71333-y

    a Immuno-TLC of extracted gangliosides from CHO-Δ Slc33a1 (Ex1-5 del) cells transfected with a plasmid encoding human V5-tagged ST8SIA1, alone or in combination with a plasmid encoding the indicated N-terminally Flag-tagged SLC33A1 variants. Mock transfected cells were used as negative control. Total gangliosides were extracted and separated by TLC followed by staining with anti-9- O -Ac-GD3 mAb M-T6004 or anti-GD3 mAb R24. Representative plates from one of three independent experiments are shown. b Flow cytometric analysis of CHO-Δ Slc33a1 (Ex1-5 del) cells transiently transfected as described in ( a ) and stained with anti-9- O -Ac-GD3 mAb M-T6004. Positive cells were gated as shown in Supplementary Fig. . Data are presented as mean ± s.d. of n = 4 independent experiments. One-way ANOVA followed by Bonferroni’s post-hoc test. **** p < 0.0001 indicates a significant difference in comparison to ST8SIA1/SLC33A1-WT expressing cells. Western blot analysis was performed to validate the expression of all Flag-tagged SLC33A1 variants. Protein bands representing full-length SLC33A1 and the C-terminally truncated variant p.Y366* are marked by an arrow. The bands marked by asterisks are already present in the lysate of mock- and ST8SIA1 -transfected cells and represent non-specific cross-reactivity of the anti-Flag antibody with endogenous cellular proteins. Actin was used as loading controls. c Cartoon representation of the cryo-EM structure of SLC33A1 (PDB ID 9M0S) . The N- and C-terminal six-helix bundles of SLC33A1 are colored in green and beige, respectively. Side chains of the residues in position 110 and 113 are shown as sticks. Dotted red lines visualize the course of the helix axes of TMH2, TMH11, and TMH12. Detail enlargements of the SLC33A1 structure highlighting the positioning of A110 ( d ) and G509 ( e ). Helices are shown as semi-transparent ribbons with selected residues shown in stick representation. Hydrogen bonds are indicated as dotted lines. For clarity, only the main chain atoms of L474 and V471 are depicted. f Schematic representation of the membrane topology of SLC33A1. Positions of patient-derived SLC33A1 mutations are indicated. Source data and exact p values are provided as a file.
    Figure Legend Snippet: a Immuno-TLC of extracted gangliosides from CHO-Δ Slc33a1 (Ex1-5 del) cells transfected with a plasmid encoding human V5-tagged ST8SIA1, alone or in combination with a plasmid encoding the indicated N-terminally Flag-tagged SLC33A1 variants. Mock transfected cells were used as negative control. Total gangliosides were extracted and separated by TLC followed by staining with anti-9- O -Ac-GD3 mAb M-T6004 or anti-GD3 mAb R24. Representative plates from one of three independent experiments are shown. b Flow cytometric analysis of CHO-Δ Slc33a1 (Ex1-5 del) cells transiently transfected as described in ( a ) and stained with anti-9- O -Ac-GD3 mAb M-T6004. Positive cells were gated as shown in Supplementary Fig. . Data are presented as mean ± s.d. of n = 4 independent experiments. One-way ANOVA followed by Bonferroni’s post-hoc test. **** p < 0.0001 indicates a significant difference in comparison to ST8SIA1/SLC33A1-WT expressing cells. Western blot analysis was performed to validate the expression of all Flag-tagged SLC33A1 variants. Protein bands representing full-length SLC33A1 and the C-terminally truncated variant p.Y366* are marked by an arrow. The bands marked by asterisks are already present in the lysate of mock- and ST8SIA1 -transfected cells and represent non-specific cross-reactivity of the anti-Flag antibody with endogenous cellular proteins. Actin was used as loading controls. c Cartoon representation of the cryo-EM structure of SLC33A1 (PDB ID 9M0S) . The N- and C-terminal six-helix bundles of SLC33A1 are colored in green and beige, respectively. Side chains of the residues in position 110 and 113 are shown as sticks. Dotted red lines visualize the course of the helix axes of TMH2, TMH11, and TMH12. Detail enlargements of the SLC33A1 structure highlighting the positioning of A110 ( d ) and G509 ( e ). Helices are shown as semi-transparent ribbons with selected residues shown in stick representation. Hydrogen bonds are indicated as dotted lines. For clarity, only the main chain atoms of L474 and V471 are depicted. f Schematic representation of the membrane topology of SLC33A1. Positions of patient-derived SLC33A1 mutations are indicated. Source data and exact p values are provided as a file.

    Techniques Used: Transfection, Plasmid Preparation, Negative Control, Staining, Comparison, Expressing, Western Blot, Variant Assay, Cryo-EM Sample Prep, Membrane, Derivative Assay

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    Western Blot:

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    Article Snippet: .. The primary antibodies (Abs) used for Western blotting included a custom-made rabbit polyclonal Ab (PAb) against the N-terminal 120 aa of PPARγ (PPARγ-N) ( 47 ), a rabbit monoclonal antibody (MAb) against the C terminus of PPARγ (PPARγ-C) (81B8; Cell Signaling Technology), a rabbit anti-RXRα MAb (ab125001; Abcam), a rabbit anti-Flag PAb (catalog number 2368; Cell Signaling Technology), a mouse anti-LCoR MAb (C6; Santa Cruz Biotechnology), and a rabbit anti-KLF6 PAb (R-173; Santa Cruz Biotechnology). .. The horseradish peroxidase (HRP)-conjugated secondary Abs used included the following: with PPARγ-N, RXRα, Flag, and KLF6 Abs, mouse anti-rabbit IgG, light chain-specific MAb (catalog number 211-032-171; Jackson ImmunoResearch); with anti-LCoR Ab (C6), mouse IgGκ binding protein-HRP (catalog number sc-516102; Santa Cruz Biotechnology); and with PPARγ-C MAb (81B8), which is not recognized by the light chain-specific Ab, affinity purified goat anti-rabbit IgG(H+L) (catalog number 111-005-144; Jackson ImmunoResearch).

    Blocking Assay:

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
    Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).

    Staining:

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
    Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).

    Purification:

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
    Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).

    Affinity Chromatography:

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
    Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).

    Cell Culture:

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
    Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).

    Incubation:

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O-acetylation in CASD1 catalysis.
    Article Snippet: .. Membranes were blocked in 3% BSA in PBS and incubated with rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.02 μgmL−1; Cell Signaling cat. 2368) overnight at 4 °C. .. Membranes werewashed and incubated for 1 h at room temperaturewith goat antirabbit IgG horseradish peroxidase (POD)-conjugate (1:20,000; SigmaAldrich cat. A6154), followed by enhanced chemiluminescence (ECL) detection using an Amersham Imager 680 (Cytiva).

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
    Article Snippet: .. Membranes were blocked in 3% BSA in PBS and incubated with rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.02 μg mL −1 ; Cell Signaling cat. 2368) overnight at 4 °C. .. Membranes were washed and incubated for 1 h at room temperature with goat anti-rabbit IgG horseradish peroxidase (POD)-conjugate (1:20,000; Sigma-Aldrich cat. A6154), followed by enhanced chemiluminescence (ECL) detection using an Amersham Imager 680 (Cytiva).



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    https://www.bioz.com/product/rabbit+anti+flag+pab/DYKDDDDK+Tag+Rabbit+mAb/10__1016_slash_j__cj__2025__10__004-110-5-9
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    ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with <t>mouse</t> <t>anti-Flag</t> mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments
    Flag Tag Rabbit Pab, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with <t>mouse</t> <t>anti-Flag</t> mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments
    Rabbit Pab, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with <t>mouse</t> <t>anti-Flag</t> mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments
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    Image Search Results


    a Immuno-TLC of extracted gangliosides from CHO-Δ Slc33a1 (Ex1-5 del) cells transfected with a plasmid encoding human V5-tagged ST8SIA1, alone or in combination with a plasmid encoding the indicated N-terminally Flag-tagged SLC33A1 variants. Mock transfected cells were used as negative control. Total gangliosides were extracted and separated by TLC followed by staining with anti-9- O -Ac-GD3 mAb M-T6004 or anti-GD3 mAb R24. Representative plates from one of three independent experiments are shown. b Flow cytometric analysis of CHO-Δ Slc33a1 (Ex1-5 del) cells transiently transfected as described in ( a ) and stained with anti-9- O -Ac-GD3 mAb M-T6004. Positive cells were gated as shown in Supplementary Fig. . Data are presented as mean ± s.d. of n = 4 independent experiments. One-way ANOVA followed by Bonferroni’s post-hoc test. **** p < 0.0001 indicates a significant difference in comparison to ST8SIA1/SLC33A1-WT expressing cells. Western blot analysis was performed to validate the expression of all Flag-tagged SLC33A1 variants. Protein bands representing full-length SLC33A1 and the C-terminally truncated variant p.Y366* are marked by an arrow. The bands marked by asterisks are already present in the lysate of mock- and ST8SIA1 -transfected cells and represent non-specific cross-reactivity of the anti-Flag antibody with endogenous cellular proteins. Actin was used as loading controls. c Cartoon representation of the cryo-EM structure of SLC33A1 (PDB ID 9M0S) . The N- and C-terminal six-helix bundles of SLC33A1 are colored in green and beige, respectively. Side chains of the residues in position 110 and 113 are shown as sticks. Dotted red lines visualize the course of the helix axes of TMH2, TMH11, and TMH12. Detail enlargements of the SLC33A1 structure highlighting the positioning of A110 ( d ) and G509 ( e ). Helices are shown as semi-transparent ribbons with selected residues shown in stick representation. Hydrogen bonds are indicated as dotted lines. For clarity, only the main chain atoms of L474 and V471 are depicted. f Schematic representation of the membrane topology of SLC33A1. Positions of patient-derived SLC33A1 mutations are indicated. Source data and exact p values are provided as a file.

    Journal: Nature Communications

    Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis

    doi: 10.1038/s41467-026-71333-y

    Figure Lengend Snippet: a Immuno-TLC of extracted gangliosides from CHO-Δ Slc33a1 (Ex1-5 del) cells transfected with a plasmid encoding human V5-tagged ST8SIA1, alone or in combination with a plasmid encoding the indicated N-terminally Flag-tagged SLC33A1 variants. Mock transfected cells were used as negative control. Total gangliosides were extracted and separated by TLC followed by staining with anti-9- O -Ac-GD3 mAb M-T6004 or anti-GD3 mAb R24. Representative plates from one of three independent experiments are shown. b Flow cytometric analysis of CHO-Δ Slc33a1 (Ex1-5 del) cells transiently transfected as described in ( a ) and stained with anti-9- O -Ac-GD3 mAb M-T6004. Positive cells were gated as shown in Supplementary Fig. . Data are presented as mean ± s.d. of n = 4 independent experiments. One-way ANOVA followed by Bonferroni’s post-hoc test. **** p < 0.0001 indicates a significant difference in comparison to ST8SIA1/SLC33A1-WT expressing cells. Western blot analysis was performed to validate the expression of all Flag-tagged SLC33A1 variants. Protein bands representing full-length SLC33A1 and the C-terminally truncated variant p.Y366* are marked by an arrow. The bands marked by asterisks are already present in the lysate of mock- and ST8SIA1 -transfected cells and represent non-specific cross-reactivity of the anti-Flag antibody with endogenous cellular proteins. Actin was used as loading controls. c Cartoon representation of the cryo-EM structure of SLC33A1 (PDB ID 9M0S) . The N- and C-terminal six-helix bundles of SLC33A1 are colored in green and beige, respectively. Side chains of the residues in position 110 and 113 are shown as sticks. Dotted red lines visualize the course of the helix axes of TMH2, TMH11, and TMH12. Detail enlargements of the SLC33A1 structure highlighting the positioning of A110 ( d ) and G509 ( e ). Helices are shown as semi-transparent ribbons with selected residues shown in stick representation. Hydrogen bonds are indicated as dotted lines. For clarity, only the main chain atoms of L474 and V471 are depicted. f Schematic representation of the membrane topology of SLC33A1. Positions of patient-derived SLC33A1 mutations are indicated. Source data and exact p values are provided as a file.

    Article Snippet: Membranes were blocked in 3% BSA in PBS and incubated with rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.02 μg mL −1 ; Cell Signaling cat. 2368) overnight at 4 °C.

    Techniques: Transfection, Plasmid Preparation, Negative Control, Staining, Comparison, Expressing, Western Blot, Variant Assay, Cryo-EM Sample Prep, Membrane, Derivative Assay

    ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with mouse anti-Flag mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

    doi: 10.1007/s00018-026-06146-9

    Figure Lengend Snippet: ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with mouse anti-Flag mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments

    Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Virus, Purification, Staining

    ENO1 prevents the binding between NP and importin α3. ( A – H ) The effect of ENO1 on the interaction between NP and importin α family members was determined by performing co-IP assays. HEK293T cells were transfected with HA-tagged NP in combination with Flag-tagged importin α1 (A, B), α3 (C, D), α5 (E, F), or α7 (G, H), together with V5-tagged ENO1 or not. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb or mouse anti-HA mAb, followed by western blotting. ( I ) A549 cells were transfected with si-Importin α3 or NC. At 24 h post-transfection, the knockdown efficiency was verified by western blotting. ( J ) Viral replication in si-Importin α3-treated A549 cells. A549 cells treated with si-Importin α3 or NC were transfected with Flag-tagged ENO1 or EV, and then infected with H1N1 virus (MOI = 0.1). At the indicated timepoints, supernatants were collected and virus titers were assessed by using TCID 50 assays. *, P < 0.05; **, P < 0.01. The data are representative of at least three independent experiments, and the mean ± SD are shown in I ( n = 3)

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

    doi: 10.1007/s00018-026-06146-9

    Figure Lengend Snippet: ENO1 prevents the binding between NP and importin α3. ( A – H ) The effect of ENO1 on the interaction between NP and importin α family members was determined by performing co-IP assays. HEK293T cells were transfected with HA-tagged NP in combination with Flag-tagged importin α1 (A, B), α3 (C, D), α5 (E, F), or α7 (G, H), together with V5-tagged ENO1 or not. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb or mouse anti-HA mAb, followed by western blotting. ( I ) A549 cells were transfected with si-Importin α3 or NC. At 24 h post-transfection, the knockdown efficiency was verified by western blotting. ( J ) Viral replication in si-Importin α3-treated A549 cells. A549 cells treated with si-Importin α3 or NC were transfected with Flag-tagged ENO1 or EV, and then infected with H1N1 virus (MOI = 0.1). At the indicated timepoints, supernatants were collected and virus titers were assessed by using TCID 50 assays. *, P < 0.05; **, P < 0.01. The data are representative of at least three independent experiments, and the mean ± SD are shown in I ( n = 3)

    Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Knockdown, Infection, Virus