Journal: Brain Communications

Article Title: Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis

doi: 10.1093/braincomms/fcad109

Figure Lengend Snippet: Immunocytochemistry of anti-FLOT1/2 antibodies (in house transfection) . HEK293 cells co-transfected with mammalian-expression vectors encoding human FLOT1 and FLOT2 using Lipofectamine 2000; double-stained with serum ( B , E ) and with commercial antibody against FLOT1 ( A , D ). MS patient’s IgG bind to co-transfected cells ( B ) and colocalize with FLOT1 ab ( C ); in contrast with the healthy control ( E ) that doesn’t show any reactivity against FLOT-1/2 antibodies ( F ).

Article Snippet: Briefly, mammalian expression vectors encoding human FLOT1 (SC319463, Origene) and FLOT2 (EX-H1820-M02, Genecopoeia) were transfected into HEK293 cells using Lipofectamine 2000.

Techniques: Immunocytochemistry, Transfection, Expressing, Staining, Control

Journal: Brain Communications

Article Title: Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis

doi: 10.1093/braincomms/fcad109

Figure Lengend Snippet: Immunoadsorption of anti-FLOT1/2 antibodies . HEK293 cells co-transfected with mammalian-expression vectors encoding human FLOT1 and FLOT2 double-stained with serum immunoadsorbed with non-transfected cells ( A ), FLOT1-transfected cells ( D ), FLOT2-transfected cells ( G ) or FLOT1 and FLOT2 co-transfected cells ( J ); and with commercial antibody against FLOT1 ( B , E , K ) or FLOT2 ( H ). Reactivity against the FLOT-1/2 complex was lost after serum pre-adsorption with HEK cells co-expressing FLOT1 and FLOT2 ( L ), but not after pre-adsorption with cells transfected with FLOT1 ( F ) or FLOT2 ( I ) alone or with non-transfected HEK cells ( C ).

Article Snippet: Briefly, mammalian expression vectors encoding human FLOT1 (SC319463, Origene) and FLOT2 (EX-H1820-M02, Genecopoeia) were transfected into HEK293 cells using Lipofectamine 2000.

Techniques: Transfection, Expressing, Staining, Adsorption

Journal: Annals of Translational Medicine

Article Title: Identifying potential therapeutic targets of Tang-Yi-Ping for the treatment of impaired glucose tolerance: a tandem mass tag-labeled quantitative proteomic analysis

doi: 10.21037/atm-21-4257

Figure Lengend Snippet: Specific information on 16 protein targets in the treatment of IGT with TYP

Article Snippet: For example, the expression levels of Rbp4 (boster, #PB0368, China) and Flot2 (boster, #PB0663, China) in the 3 groups were calculated with GAPDH (boster, #BM1623, China) as an internal reference.

Techniques:

Journal: Annals of Translational Medicine

Article Title: Identifying potential therapeutic targets of Tang-Yi-Ping for the treatment of impaired glucose tolerance: a tandem mass tag-labeled quantitative proteomic analysis

doi: 10.21037/atm-21-4257

Figure Lengend Snippet: Relative expression levels of Rbp4 and Flot2 proteins in pancreatic samples of the 3 groups. Rbp4: *, P<0.05, vs. Control; #, P<0.05, vs. IGT. Flot: *, P<0.05, vs. Control; #, P<0.05, vs. IGT. IGT, impaired glucose tolerance.

Article Snippet: For example, the expression levels of Rbp4 (boster, #PB0368, China) and Flot2 (boster, #PB0663, China) in the 3 groups were calculated with GAPDH (boster, #BM1623, China) as an internal reference.

Techniques: Expressing, Control

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A and B ) Tumor volume in Flot2 +/+ or Flot2 –/– mice injected with B16F10 ( A ) or MC38 ( B ) ( n = 6–7 per group). ( C – J ) Flow cytometric analysis of tumor-infiltrating lymphocytes (TILs) in B16F10 tumor–bearing Flot2 +/+ or Flot2 –/– mice. Representative plots ( C , E , and H ) are shown. TCRβ + ( D ), TCRβ + CD4 + ( F ), and TCRβ + CD8 + ( G ) populations within 7AAD – CD45 + population, and Ki67 + populations among 7AAD – CD45 + TCRβ + CD4 + CD44 + CD62L – population ( I ) or 7AAD – CD45 + TCRβ + CD8 + CD44 + CD62L – population ( J ), are depicted. ( K ) Splenocytes from B16F10 tumor–bearing Flot2 +/+ or Flot2 –/– mice were stimulated with 1 μg/mL of TRP-2 melanoma peptide for 24 hours and assayed for antigen-specific reactivity using an IFN-γ ELISPOT assay. Data are representative of 2 independent experiments. Data were analyzed by unpaired t test ( D , F , G , and I – K ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and B ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Injection, Enzyme-linked Immunospot, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A and B ) Tumor volume in Flot2 WT or Flot2 CD4 mice injected with B16F10 ( A ; n = 10 per group) or MC38 ( B , n = 9 for Flot2 WT and n = 14 for Flot2 CD4 ). ( C – H ) Flow cytometric analysis of TILs. Representative plots ( C and F ) are shown. TCRβ + CD4 + ( D ) and TCRβ + CD8 + ( E ) populations among 7AAD – CD45.2 + population and Ki67 + populations among 7AAD – CD45.2 + TCRβ + CD4 + population ( G ) or 7AAD – CD45.2 + TCRβ + CD8 + population ( H ) in B16F10-bearing Flot2 WT or Flot2 CD4 mice are presented. ( I – N ) Flow cytometric analysis of tumor-draining lymph nodes (dLNs). Representative plots ( I and L ) are displayed. CD44 + IFN-γ + ( J ) and CD44 + TNF-α + ( K ) populations among 7AAD – CD45.2 + TCRβ + CD4 + population and CD44 + IFN-γ + ( M ) and CD44 + TNF-α + ( N ) among 7AAD – CD45.2 + TCRβ + CD8 + population in B16F10-bearing Flot2 WT or Flot2 CD4 mice are indicated. Data are representative of 2 independent experiments ( A – N ). Data were analyzed by unpaired t test ( D , E , G , H , J , K , M , and N ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and B ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Injection, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A ) Weight loss in Flot2 +/+ or Flot2 –/– mice following Listeria monocytogenes infection (5,000 CFU per mouse) is presented as the mean percentage of initial weight ( n = 4–5 per group). ( B – G ) Flow cytometric analysis of splenic T cells from Listeria -infected Flot2 +/+ or Flot2 –/– mice. Representative plots ( B and E ) are provided. CD44 + Ki67 + ( C ) and TNF-α + ( D ) populations within viable CD45 + TCRβ + CD4 + population and CD44 + Ki67 + ( F ) and TNF-α + ( G ) populations within viable CD45 + TCRβ + CD8 + population are shown. ( H ) Weight loss in Flot2 WT or Flot2 CD4 mice following L. monocytogenes infection (5,000 CFU per mouse) is presented as the mean percentage of initial weight ( n = 5–6 per group). ( I ) Splenic CD4 + and CD8 + T cells numbers in infected Flot2 WT or Flot2 CD4 mice are depicted. ( J – Q ) Flow cytometric analysis of splenic T cells from L. monocytogenes –infected Flot2 WT or Flot2 CD4 mice. Representative plots ( J , L, and O ) are shown. TNF-α + IFN-γ + IL-2 + ( K ), CD44 + Ki67 + ( M ), and TNF-α + ( N ) populations within viable CD45 + TCRβ + CD4 + population and CD44 + Ki67 + ( P ) and TNF-α + ( Q ) populations within viable CD45 + TCRβ + CD8 + population are shown. Data are representative of 2 independent experiments ( A – Q ). Data were analyzed by unpaired t test ( C , D , F, G, I , K , M , N , P , and Q ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and H ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Infection, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A – C ) Naive CD4 + T cells were stimulated in vitro for 3 hours ( B ) or 24 hours ( C ) with varying doses of plate-bound αCD3, alongside a fixed dose of soluble αCD28 (1 μg/mL), followed by FACS. Representative plots ( A ) show Nur77 + ( B ) and CD69 + ( C ) populations within viable TCRβ + CD4 + population. ( D – G ) Western blot of the phosphorylation of TCR signaling molecules in naive CD4 + T cells stimulated with varying doses of plate-bound αCD3 for 3 minutes. Representative blots ( D ) and quantifications of pZAP70 ( E ), pERK1/2 ( F ), and pLck (Y505) ( G ), normalized to their respective total protein levels (ZAP70, ERK1/2, and Lck), are shown. ( H and I ) scRNA-Seq was performed on naive CD4 + T cells following a 3-hour stimulation with varying concentrations of plate-bound αCD3. A fixed dose of soluble αCD28 (1 μg/mL) was provided under the conditions of 0.25 or 1 μg/mL of plate-bound αCD3. Unsupervised T cell clusters were annotated as 5 distinguishable functional states on a UMAP plot ( H ), and the effect of stimulation dose was projected onto the UMAP ( I ). ( J ) Distribution of Flot2 WT or Flot2 CD4 CD4 + T cells in each cluster. Red arrows indicate the priming clusters for each genotype. ( K ) Occupancy of Flot2 WT or Flot2 CD4 genotype in each T cell cluster, analyzed by total or each stimulatory condition. ( L ) Volcano plots of spliced RNA in naive, priming, and activated clusters. ( M ) Expression level of spliced RNA related to cellular proliferation in the naive cluster. Data are pooled from 3 ( A – C ), 5 ( E ), 8 ( F ), or 7 ( G ) independent experiments. Data were analyzed by 1-way ANOVA followed with Šidák’s multiple-comparison tests ( B , C , and E – G ), χ 2 analysis ( K ), or Wilcoxon ranked-sum test ( M ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: In Vitro, Western Blot, Phospho-proteomics, Functional Assay, Expressing, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A – H ) dSTORM analysis of TCR nanoclustering in Flot2 WT and Flot2 CD4 naive CD4 + T cells. Clustering images of CD3ε molecules ( A and B ) or TCRβ molecules ( E and F ) are shown. Number of clusters and voxel of CD3ε ( C and D ) or TCRβ ( G and H ) molecules after quantification using Huygens Cluster Analyzer are depicted. Scale bars: 1 μm. Data are representative of 4 ( A – D ) or 2 ( E – H ) independent experiments. Data were analyzed by unpaired t test ( C , D , G , and H ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques:

Journal: Histochemistry and cell biology

Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis

doi: 10.1007/s00418-012-1040-2

Figure Lengend Snippet: FLOT1 and FLOT2 are expressed in human placental homogenates. (A) Equal amounts of protein from fractions generated during the preparation of small cationic colloidal silica-coated ST microvillous fractions (Robinson et al., 2009b) were resolved by gel electrophoresis, transferred to nitrocellulose membranes, and probed with antibodies directed against either FLOT1 or FLOT2. These fractions were: crude tissue homogenate (CTH), filtered crude tissue homogenate (FCTH), crude placental supernatant (CPS), crude placental pellet (CPP), Histodenz fractions (0–50%, 50–55%), and the pelleted plasma membrane (65% PPM). Note that neither FLOT1 nor FLOT2 was enriched in the PPM fraction. (B) Immunoblotting was used to confirm the expression of both flotillins in three additional term human placenta extracts prepared following lysis in buffer containing n-octyl β-D-glucopyranoside (see “Materials and methods”).

Article Snippet: The following antibodies were used for immunoblotting and immunolabeling experiments, as indicated in the text and figure legends: mouse anti-FLOT1 (610820, BD Biosciences), mouse anti-FLOT2 (610383, BD Biosciences), rabbit anti-FLOT1 (HPA001393, Sigma-Aldrich), rabbit anti-FLOT2 (F1680, Sigma-Aldrich), rabbit anti-FLOT2 (F1805, Sigma-Aldrich), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon International), mouse anti-dysferlin (DYSF; Ham1/7B6, Vector Laboratories), mouse monoclonal anti-E-cadherin (E-cad, 610181, BD Biosciences), and mouse anti-serine peptidase inhibitor, Kunitz type 1 (SPINT1, clone C76/18) ( Kataoka et al., 2000b ; Kataoka et al., 2000a ; Shimomura et al., 1999 ).

Techniques: Generated, Nucleic Acid Electrophoresis, Western Blot, Expressing, Lysis

Journal: Histochemistry and cell biology

Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis

doi: 10.1007/s00418-012-1040-2

Figure Lengend Snippet: Distribution of FLOT1 and FLOT2 proteins in term human placental villi. (A–D) Representative photomicrographs of conventional cryostat sections (5–6 μm) of placental villi labeled with anti-FLOT1 (HPA001393; red in A) and anti-FLOT2 (1608; red C) antibodies and imaged using laser scanning confocal microscopy. The intensities of these images were adjusted to provide maximal contrast. Panels B and D are corresponding non-confocal differential interference contrast (DIC) micrographs on which DAPI staining (blue) is superimposed. Prominent flotillin labeling (red) is observed in cytotrophoblasts (CTs) and/or basal regions of syncytiotrophoblast (ST), and in fetal capillary endothelial cells (*, lumen of fetal capillary). Arrowheads indicate surface of ST; double arrows indicate flotillin labeling in ST; white single arrows indicate labeling along the basal surface of the ST and/or in CTs; black arrows indicate labeling in endothelial cells. (E, F) Fluorescence intensity measurements for FLOT1 (E) and FLOT2 (F) labeling taken within fetal capillary endothelial cells (EC) of intermediate and terminal villi, and at three levels of the villous tree: terminal villi (TV), intermediate villi (IV), and stem villi (SV). In our analysis, we found that the intensity measurements for FLOT1 EC labeling in intermediate and terminal villi were equivalent, so the data were pooled. This was also the case for FLOT2 EC labeling. Note that the differences in the absolute intensity measurements between FLOT1 and FLOT2 may represent differences in antigen-antibody interactions, and should not be interpreted as differences in relative abundance between these two proteins. Data are mean ± SD from 3 separate term placental specimens; *, P < 0.05 vs. SV; **, P < 0.01 vs. SV (ANOVA with Bonferroni multiple comparisons). Scale bar = 20 μm

Article Snippet: The following antibodies were used for immunoblotting and immunolabeling experiments, as indicated in the text and figure legends: mouse anti-FLOT1 (610820, BD Biosciences), mouse anti-FLOT2 (610383, BD Biosciences), rabbit anti-FLOT1 (HPA001393, Sigma-Aldrich), rabbit anti-FLOT2 (F1680, Sigma-Aldrich), rabbit anti-FLOT2 (F1805, Sigma-Aldrich), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon International), mouse anti-dysferlin (DYSF; Ham1/7B6, Vector Laboratories), mouse monoclonal anti-E-cadherin (E-cad, 610181, BD Biosciences), and mouse anti-serine peptidase inhibitor, Kunitz type 1 (SPINT1, clone C76/18) ( Kataoka et al., 2000b ; Kataoka et al., 2000a ; Shimomura et al., 1999 ).

Techniques: Labeling, Confocal Microscopy, Staining, Fluorescence

Journal: Histochemistry and cell biology

Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis

doi: 10.1007/s00418-012-1040-2

Figure Lengend Snippet: Flotillin expression is downregulated and changes distribution in BeWo cells following foskolin-induced fusion. (A) Representative immunoblots of BeWo cells treated with 20 μM forskolin from 0–72 h. Note that the immunoreactivity of FLOT1 and FLOT2 decreases with forskolin treatment, whereas that of dysferlin (DYSF) increases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in three separate experiments. (B, C) Mononuclear BeWo cells immunolabeled with antibodies against FLOT1 (HPA001393; red in B) and FLOT2 (1608; red in C), respectively. Double arrows indicate areas of flotillin labeling along cellular boundaries; wide arrows denote areas of periuclear staining in individual cells. (D, E) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using antibodies generated against E-cadherin (E-cad, green) and either FLOT1 (red in D) or FLOT2 (red in E), as above. Wide arrows indicate flotillin-like immunoreactivity in crescent-shaped structures in close proximity to clusters of nuclei; arrowhead in E denotes an area of Ecad labeling that has become irregular following cell-cell fusion. (F) Cryosection (5–6 μm) of term placental villus co-immunolabeled using antibodies generated against dysferlin (DYSF, green) and FLOT2 (1608; red) and imaged using confocal microscopy. Single arrows indicate DYSF labeling at the apical regions of the ST; double arrows indicate FLOT2 labeling. (G) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using anti-DYSF (green) and anti-FLOT2 (red) antibodies. Arrowheads denote co-labeling in crescent-shaped structures around clusters of nuclei; the asterisk indicates DYSF immunoreactivity in fused cells. Note that non-fused cells lack DYSF labeling. All scale bars = 20 μm

Article Snippet: The following antibodies were used for immunoblotting and immunolabeling experiments, as indicated in the text and figure legends: mouse anti-FLOT1 (610820, BD Biosciences), mouse anti-FLOT2 (610383, BD Biosciences), rabbit anti-FLOT1 (HPA001393, Sigma-Aldrich), rabbit anti-FLOT2 (F1680, Sigma-Aldrich), rabbit anti-FLOT2 (F1805, Sigma-Aldrich), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon International), mouse anti-dysferlin (DYSF; Ham1/7B6, Vector Laboratories), mouse monoclonal anti-E-cadherin (E-cad, 610181, BD Biosciences), and mouse anti-serine peptidase inhibitor, Kunitz type 1 (SPINT1, clone C76/18) ( Kataoka et al., 2000b ; Kataoka et al., 2000a ; Shimomura et al., 1999 ).

Techniques: Expressing, Western Blot, Immunolabeling, Labeling, Staining, Generated, Confocal Microscopy

Journal: Histochemistry and cell biology

Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis

doi: 10.1007/s00418-012-1040-2

Figure Lengend Snippet: Flotillins contribute to endocytosis in BeWo cells. (A) Flotillins co-localize with a fluorescent marker of fluid phase-pinocytosis in endocytosis assays. Mononuclear BeWo cells were incubated continuously in the presence of 1 mg/ml of lucifer yellow (LY-CH, green) for 15 min prior to fixation. Following permeabilization, the cells were immunolabeled with anti-FLOT2 antibodies (red) and imaged using confocal microscopy. In the representative micrograph taken from a single optical section, areas of co-localization between the LY-CH cargo and FLOT2 (yellow in the merged images, indicated by arrows) are observed. Similar results were obtained when the cells were labeled using antibodies generated against FLOT1 (not shown). Scale bar = 10 μm. (B) Immunoblots of BeWo cell lines stably transuced with shRNA targeting FLOT1, FLOT2, or both. Control cells were tranduced with Non-Target Control Transduction Particles (LV CTRL) as described in “Materials and methods”. (C) Flotillin knockdown decreases uptake of fluorescently-labeled cholera toxin B subunit (CTB-594). BeWo cells varying in flotillin expression levels were incubated with 20 μg/ml CTB-594 at 4°C for 30 min, then warmed to 37°C and endocytosis was allowed to proceed in media without labeled ligand for 15 min. On average, cells deficient in flotillin expression exhibited decreased CTB-549 based on average fluorescence intensity measurements. Data are mean ± SD from two separate experiments. Different letters indicate significant differences at P < 0.05 (ANOVA with Bonferroni multiple comparisons)

Article Snippet: The following antibodies were used for immunoblotting and immunolabeling experiments, as indicated in the text and figure legends: mouse anti-FLOT1 (610820, BD Biosciences), mouse anti-FLOT2 (610383, BD Biosciences), rabbit anti-FLOT1 (HPA001393, Sigma-Aldrich), rabbit anti-FLOT2 (F1680, Sigma-Aldrich), rabbit anti-FLOT2 (F1805, Sigma-Aldrich), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon International), mouse anti-dysferlin (DYSF; Ham1/7B6, Vector Laboratories), mouse monoclonal anti-E-cadherin (E-cad, 610181, BD Biosciences), and mouse anti-serine peptidase inhibitor, Kunitz type 1 (SPINT1, clone C76/18) ( Kataoka et al., 2000b ; Kataoka et al., 2000a ; Shimomura et al., 1999 ).

Techniques: Marker, Incubation, Immunolabeling, Confocal Microscopy, Labeling, Generated, Western Blot, Stable Transfection, shRNA, Transduction, Expressing, Fluorescence