3xflag epitope tag  (5 PRIME)

Journal: The Journal of Cell Biology

Article Title: Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1

doi: 10.1083/jcb.202011078

Figure Lengend Snippet: Validation of a covalent, reversible cross-linking of Ire1 homodimers via disulfide bridges. (A) A cross-linking experiment using CuSO 4 was performed with microsomes prepared from cells expressing a HA-tagged variant of Ire1 from endogenous locus ( IRE1 3xHA-GFP ) and a Flag-tagged variant ( IRE1 3xFlag-GFP ) from a CEN-based plasmid. A yeast culture in selective medium without leucine was inoculated to an OD 600 of 0.2 from a stationary overnight culture and cultivated at 30°C until an OD 600 of 0.7 was reached. The cells were either stressed with 2 mM DTT or left untreated and were further cultivated for 1 h. 80 OD 600 equivalents from these cultures were harvested by centrifugation. Microsomal membranes were isolated by differential centrifugation. Microsomes prepared from cells expressing only one of the two tagged variants of Ire1 served as controls. Both constructs contained a single cysteine in the TMH region at the position 552 (C552). After incubation of the microsomes with 10 mM CuSO 4 on ice for 5 min, the cross-linking reaction was stopped by the addition of NEM in a final concentration of 111 mM and EDTA in a final concentration of 50 mM. The microsomes were then solubilized using 2% Triton X-100 and subjected to an IP using anti-Flag beads. Both the input and IP samples were analyzed by immunoblotting using anti-Flag and anti-HA antibodies. (B) The reversibility of the cysteine-mediated cross-link was validated using the indicated F544C variant of Ire13xHA-GFP. The cross-link was induced by CuSO 4 in microsomes prepared from cells stressed with TM as described in . The cross-link was reverted by treating the sample with 90 mM DTT and incubating at 70° and 95° as indicated. The monomeric and dimeric species of Ire1 are indicated by symbols.

Article Snippet: The 3xHA epitope tag in the knock-in construct was replaced by a 3xFlag epitope tag using the Q5 site-directed mutagenesis kit (NEB).

Techniques: Biomarker Discovery, Expressing, Variant Assay, Plasmid Preparation, Centrifugation, Isolation, Construct, Incubation, Concentration Assay, Western Blot

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a-c) mRNA expression levels of GLUT and SGLT transporter isoforms in BMDMs (a), THP-1 cells (b), and Raw 264.7 cells (c) in unstimulated macrophages (white) and after treatment with classic M1 (red) or alternative M2 (blue) polarization stimuli for 24 hours. Expression normalized to β-actin (ACTB) expression (d) mRNA expression levels of M1 (Nos2, Tnfa and Il1b) and M2 (Arg1, Retnla and Chil3l3) markers in WT (n=12) and GLUT1 KO (n=12) BMDMs after the indicated polarization stimuli. (e) mRNA expression levels of M1 and M2 markers in WT (n=12) and GLUT3 KO (n=12) BMDMs after the indicated polarization stimuli. (f) 2-Deoxy-D-glucose uptake in WT, GLUT1 KO, and GLUT3 KO BMDMs after the indicated polarization stimuli. Data shown as mean ± SEM. P values were calculated by two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a) Scheme for calcipotriol (MC903) induced dermatitis. Calcipotriol (1.125 nmol) in ethanol was applied to the left ear and shaved back of the indicated mice for 13 days. (b) Representative photos after calcipotriol administration in WT, GLUT3 KO and GLUT1 KO mice at day 8. (c) Hematoxylin and eosin stained sections of mouse skin treated with calcipotriol analyzed at day 13 in WT, GLUT3 KO, and GLUT1 KO mice. (d) Thickness of calcipotriol-treated ear and back in WT (n=11 for ear and n=6 for back), GLUT3 KO (n=12 for ear and n=8 for back), and GLUT1 KO (n=10 for ear and n=4 for back) mice. (e-g) mRNA expression levels in calcipotriol-treated ear in WT (n=3), GLUT3 KO (n=4), and GLUT1 KO (n=3) mice. Pan-macrophage marker (F4/80), M1 markers (Nos2 and Tnfa) (e), M2 markers (Arg1, Mrc1 and Retnla) (f), and Th2 cytokines (IL-4, IL-13, and IL-31) (g) were observed. Data shown as mean ± SEM. P values were calculated by two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Staining, Expressing, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a) Scheme for splinted wound healing model. Wounds on the shaved back of WT, GLUT1, and GLUT3 KO mice were generated by 3.0 mm punch biopsy, splinted, and wound healing was measured every two days. (b) Representative photos of wound site in WT, GLUT3 KO and GLUT1 KO mice at 6 days after injury. (c) Measurements of wound diameter on day 6 in WT (n=12), GLUT3 KO (n=12) and GLUT1 KO (n=7) mice. (d-e) mRNA expression levels of F4/80, Nos2 and Tnfa in WT (n=3), GLUT3 KO (n=3) and GLUT1 KO (n=3) mice. (f) Representative immunofluorescence stains of Arg1 (green), F4/80 (red) and DAPI (blue) in the wound site at 6 days after injury. Scale bar = 50 μm. ( g ) Statistical analysis of M2 macrophages at wound sites. The number of F4/80 + Arg1 + cells was normalized to the number of F4/80 + cells. (h-i) mRNA expression levels of tissueremodeling related markers (Tgfb, Acta2 and Col3a1) (h) and angiogenesis markers (Vegfa, Tek and Cxcr3) (i). Data shown as mean ± SEM. P values were calculated by two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Generated, Expressing, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a) Western blot analysis of the expression of phospho-STAT1 (Y701) and total STAT1 in WT and GLUT1 KO BMDMs after LPS and IFNγ stimulation (upper panel). The expression of phospho-STAT6 (Y641) and total STAT6 in WT and GLUT3 KO BMDMs after IL-4 stimulation (lower panel). (b ) Representative Western blot showing IL-4–mediated protein expression of pSTAT6 and STAT6 in WT and GLUT3 KO BMDMs. (c-d) Western blot analysis of the expression of pSTAT6 and STAT6 after knockdown of endogenous GLUT3 in THP-1 cells and RAW 264.7 cells using sh-RNA and si-RNA, respectively. (e) The expression of phospho-JAK1 (Y1034/1035) and total JAK1 was analyzed in WT and GLUT3 KO BMDMs after IL-4 stimulation. (f) Expression of pSTAT6 and STAT6 after overexpression of wild type GLUT3 or GLUT3 R331W mutant and knockdown of endogenous GLUT3 in THP-1 cells. (g) The expression of pSTAT6 and total STAT6 was analyzed in THP-1 cells after treatment with each concentration of G3iA and IL-4.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Mutagenesis, Concentration Assay

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a) Western blot analysis of GLUT3 mutant alleles in Rat2 fibroblasts after lentiviral transduction of the indicated plasmid. HSP90, loading control. (b) Representative immunofluorescence stains of Flag-GLUT3 (green) and DAPI (blue) in vector, GLUT3 WT, GLUT3 sh1sh3 resistant, and GLUT3 R331W expressing Rat2 fibroblasts. (c) 2-Deoxy-D-glucose uptake in Rat2 fibroblasts expressing each plasmid. Data shown as mean ± SEM. P values were calculated by one-way ANOVA with Dunnett tests. ****P < 0.0001.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Western Blot, Mutagenesis, Transduction, Plasmid Preparation, Control, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a) Western blot of GLUT1 and GLUT3 in membrane, cytosol, nuclear, chromatin, and cytoskeletal fraction from THP-1 cells. (b) Representative immunofluorescence stains of GLUT1 (upper panel, green), GLUT3 (lower panel, green), EEA1 (red), and DAPI (blue) in THP-1 cells. Scale bar = 10 μm. (c-e) Western blot analysis of the expression of GLUT3, GLUT1, pSTAT6 and STAT6 in the isolated plasma membrane (PM) and endosome fraction from WT and GLUT3 KO BMDMs (c), THP-1 cells (d) and RAW 264.7 cells (e). Na + /K + -ATPase and EEA1 were used as fractionation controls for the plasma membrane and endosome, respectively. (f) Western blot analysis of the expression of pSTAT6 and STAT6 in THP-1 cells in the presence or absence of IL-4 and dynasore.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Western Blot, Membrane, Immunofluorescence, Expressing, Isolation, Clinical Proteomics, Fractionation

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a) Interaction between GLUT3 and Ras in WT BMDMs. GLUT3 was immunoprecipitated from the cell lysates and Ras (D2C1 Rabbit mAb recognizing N-Ras and K-Ras) was detected by Western blot. (b) HEK293T cells were transfected with the indicated GLUT allele, and GLUT1 (Fisher Scientific, MA1-37783) or GLUT3 (Abcam, ab15311) were immunoprecipitated. Ras was detected by Western blot. Normal mouse IgG for GLUT1 and a normal rabbit IgG for GLUT3 were used as IP controls. (c) HEK293T cells were transfected with the indicated GLUT3 allele (see ) and GLUT3 alleles were Flag immunoprecipitated; Ras was detected by Western blot. IgG indicates a normal mouse IgG as IP control. (d) Expression of pSTAT6 and STAT6 after expression of indicated GLUT3 allele and knockdown of endogenous GLUT3 in THP-1 cells. (e, f) Protein expression of phospho-PAK, total PAK, phospho-cofilin (S3) and total cofilin in the presence or absence of IL-4 in WT, GLUT3 KO BMDMs (c) and THP-1 cells transfected with sh-control or sh-GLUT3 plasmid (d). (g, h) Western blot analysis of the expression of IL4Rα and Cγ chain in the isolated plasma membrane (PM) and endosomal fraction from WT, GLUT3 KO BMDMs (f) and THP-1 cells lentivirally transduced with sh-Con or sh-GLUT3 plasmid (g). Na + /K + -ATPase and EEA1 were used as fractionation controls.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Immunoprecipitation, Western Blot, Transfection, Control, Expressing, Knockdown, Plasmid Preparation, Isolation, Clinical Proteomics, Membrane, Transduction, Fractionation

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: Alleles were amino-terminally tagged with a 3xFlag epitope tag. Dashed boxes indicated predicted transmembrane (TM) domains.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques:

Journal: bioRxiv

Article Title: Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

doi: 10.1101/2022.11.09.515804

Figure Lengend Snippet: (a) Western blot for IL4Rα and Cγ chain in WT and GLUT3 KO BMDMs. GAPDH, loading control. (b) mRNA levels of IL4Rα and Cγ chain transcripts in WT and GLUT3 KO BMDMs. (c) Western blot for IL4Rα and Cγ chain in WT and GLUT3 KO IL4Rα and Cγ chain in THP-1 cells transduced with sh-Con or sh-GLUT3 plasmid. GAPDH, loading control. (d) mRNA levels of IL4Rα and Cγ chain transcripts in THP-1 cells transduced with sh-Con or sh-GLUT3 plasmid.

Article Snippet: An amino-terminal 3xFlag epitope tagged human GLUT3 was generated by PCR (Addgene #72877) (Supplementary Table 1).

Techniques: Western Blot, Control, Transduction, Plasmid Preparation