Review

dna constructs expressing gfp (Addgene inc)

Addgene inc is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Addgene inc dna constructs expressing gfp

INF2 R218Q induces the formation of multipolar spindles in MDCK cells. A Percentage of Cherry and INF2 R218Q cells in mitosis after 48 h of expression. More than 1000 cells were analyzed for each experimental condition in five independent experiments. B Distribution of Cherry and INF2 R218Q cells across different phases of the cell cycle. More than 200 cells were analyzed for each experimental condition, four independent experiments. C Percentage of mitotic Cherry and INF2 R218Q cells displaying multipolar spindles. 68 Cherry cells and 152 INF2 R218Q cells were examined; four independent experiments. D Images of INF2 R218Q mitotic cells stained for centrin, α-tubulin and γ-tubulin. Nuclei were visualized using DAPI. E Classification of INF2 R218Q mitotic cells based on the number of MTOCs expressed as percentage of total cells. MTOCs were identified by γ-tubulin and α-tubulin staining. Over 200 cells were examined; four independent experiments. F Top: Schematic of centriole arrangements. Bottom: centriole arrangement, as visualized with centrin, in INF2 R218Q mitotic cells categorized as clustered in two centrosomes (2 + 2), disengaged in one (2 + 1 + 1) or both (1 + 1 + 1 + 1) centrosomes, or with an abnormal number of centrioles, presented as a percentage of total cells. More than 170 cells were examined; three independent experiments. G Top panel: schematic of ER invasion of the spindle space in cells expressing pathogenic INF2. Bottom panels: INF2 L76P cells stably expressing <t>GFP-sec61</t> and stained with <t>SiR-DNA</t> analyzed by videomicroscopy during mitosis. The arrowheads indicate regions of the mitotic spindle invaded by ER membranes. H The graph shows the percentage of wt INF2 and INF2 R218Q cells with the spindle space invaded by ER membranes. More than 80 cells were analyzed; three independent experiments. I Videomicroscopic analysis of INF2 R218Q cells stably expressing GFP-sec61 and stained with SiR-DNA during formation of multiple micronuclei. Scale bars, 5 μm. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Dna Constructs Expressing Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dna constructs expressing gfp/product/Addgene inc
Average 93 stars, based on 6 article reviews

dna constructs expressing gfp - by Bioz Stars, 2026-06

93/100 stars

Images

1) Product Images from "INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death"

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

Journal: Cellular and Molecular Life Sciences: CMLS

doi: 10.1007/s00018-024-05323-y

Figure Legend Snippet: INF2 R218Q induces the formation of multipolar spindles in MDCK cells. A Percentage of Cherry and INF2 R218Q cells in mitosis after 48 h of expression. More than 1000 cells were analyzed for each experimental condition in five independent experiments. B Distribution of Cherry and INF2 R218Q cells across different phases of the cell cycle. More than 200 cells were analyzed for each experimental condition, four independent experiments. C Percentage of mitotic Cherry and INF2 R218Q cells displaying multipolar spindles. 68 Cherry cells and 152 INF2 R218Q cells were examined; four independent experiments. D Images of INF2 R218Q mitotic cells stained for centrin, α-tubulin and γ-tubulin. Nuclei were visualized using DAPI. E Classification of INF2 R218Q mitotic cells based on the number of MTOCs expressed as percentage of total cells. MTOCs were identified by γ-tubulin and α-tubulin staining. Over 200 cells were examined; four independent experiments. F Top: Schematic of centriole arrangements. Bottom: centriole arrangement, as visualized with centrin, in INF2 R218Q mitotic cells categorized as clustered in two centrosomes (2 + 2), disengaged in one (2 + 1 + 1) or both (1 + 1 + 1 + 1) centrosomes, or with an abnormal number of centrioles, presented as a percentage of total cells. More than 170 cells were examined; three independent experiments. G Top panel: schematic of ER invasion of the spindle space in cells expressing pathogenic INF2. Bottom panels: INF2 L76P cells stably expressing GFP-sec61 and stained with SiR-DNA analyzed by videomicroscopy during mitosis. The arrowheads indicate regions of the mitotic spindle invaded by ER membranes. H The graph shows the percentage of wt INF2 and INF2 R218Q cells with the spindle space invaded by ER membranes. More than 80 cells were analyzed; three independent experiments. I Videomicroscopic analysis of INF2 R218Q cells stably expressing GFP-sec61 and stained with SiR-DNA during formation of multiple micronuclei. Scale bars, 5 μm. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Techniques Used: Expressing, Staining, Stable Transfection

INF2 R218Q induces nuclear accumulation of p53 and activation of caspase-3 in MDCK cells. A The graph depicts the time from prophase initiation to the end of cell division, as determined by time-lapse analysis, in control cells (n = 63) and in cells expressing pathogenic INF2 ending with mild (n = 48 cells) and severe (n = 36 cells) nuclear defects; three independent experiments were performed. MDCK cells stably expressing H2B-GFP were used in this experiment. B Immunoblot analysis of p53 in wt INF2 and INF2 R218Q cells. GAPDH was used as loading control. C Quantification of p53 levels in wt INF2 and R218Q cells. Five independent experiments were performed. D Image of p53 staining in an equatorial plane of wt INF2 and INF2 R218 cells. Cells were stained with anti-Cherry antibodies to help visualization of the exogenous proteins. E Percentage of cells with nuclear p53, considering staining in the main nucleus or micronuclei as positive. Over 350 cells were examined; three independent experiments. F Images of cleaved caspase-3 staining in apoptotic INF2 R218Q cells (top) and normal cells (bottom). Nuclei were visualized with DAPI. G Percentage of Cherry, wt INF2 and INF2 R218Q cells positive for cleaved caspase-3 after 48 h of expression. More than 700 cells were examined per experimental condition; three independent experiments. H The percentage of cells with normal, mild or severe nuclear morphology or in mitosis that died between 55 and 72 h of INF2 R218Q expression was determined by time-lapse microscopy in cells labeled with SiR-DNA. 130 cells were analyzed; three independent experiments. I Videomicroscopic analysis of a mitotic (left panels) and an INF2 R218Q cell with a severe phenotype (right panels) undergoing death and detachment from the substrate. Arrowheads indicate the dying cell. Nuclei were stained with SiR-DNA. DIC, differential interference contrast microscopy. Scale bars, 10 μm ( D , F ), 250 μm ( I ). n.s., not significant; **, p < 0.01; ***, p < 0.001

Figure Legend Snippet: INF2 R218Q induces nuclear accumulation of p53 and activation of caspase-3 in MDCK cells. A The graph depicts the time from prophase initiation to the end of cell division, as determined by time-lapse analysis, in control cells (n = 63) and in cells expressing pathogenic INF2 ending with mild (n = 48 cells) and severe (n = 36 cells) nuclear defects; three independent experiments were performed. MDCK cells stably expressing H2B-GFP were used in this experiment. B Immunoblot analysis of p53 in wt INF2 and INF2 R218Q cells. GAPDH was used as loading control. C Quantification of p53 levels in wt INF2 and R218Q cells. Five independent experiments were performed. D Image of p53 staining in an equatorial plane of wt INF2 and INF2 R218 cells. Cells were stained with anti-Cherry antibodies to help visualization of the exogenous proteins. E Percentage of cells with nuclear p53, considering staining in the main nucleus or micronuclei as positive. Over 350 cells were examined; three independent experiments. F Images of cleaved caspase-3 staining in apoptotic INF2 R218Q cells (top) and normal cells (bottom). Nuclei were visualized with DAPI. G Percentage of Cherry, wt INF2 and INF2 R218Q cells positive for cleaved caspase-3 after 48 h of expression. More than 700 cells were examined per experimental condition; three independent experiments. H The percentage of cells with normal, mild or severe nuclear morphology or in mitosis that died between 55 and 72 h of INF2 R218Q expression was determined by time-lapse microscopy in cells labeled with SiR-DNA. 130 cells were analyzed; three independent experiments. I Videomicroscopic analysis of a mitotic (left panels) and an INF2 R218Q cell with a severe phenotype (right panels) undergoing death and detachment from the substrate. Arrowheads indicate the dying cell. Nuclei were stained with SiR-DNA. DIC, differential interference contrast microscopy. Scale bars, 10 μm ( D , F ), 250 μm ( I ). n.s., not significant; **, p < 0.01; ***, p < 0.001

Techniques Used: Activation Assay, Control, Expressing, Stable Transfection, Western Blot, Staining, Time-lapse Microscopy, Labeling, Microscopy

INF2 R218Q requires integrity of its actin polymerization activity to produce nuclear abnormalities. A Dynamics of wt INF2 during mitosis in MDCK cells expressing GFP-tubulin. Cells were stained with SiR-DNA. B Images of INF2 KO MDCK cells expressing INF2 R218Q and R218Q IA. ( C ) Box plots showing the intensity of the perinuclear F-actin and Cherry corresponding to INF2-1 R218Q and INF2-1 R218Q IA MDCK KO cells. More than 150 cells were examined; three independent experiments. D Percentage of MDCK KO cells expressing INF2-1 R218Q IA and INF2-2 R218Q IA exhibiting mild or severe nuclear phenotypes. More than 350 cells were examined; three independent experiments. E Images of an equatorial plane of INF2 KO MDCK cells expressing INF2 A149D and INF2 3LA. F Percentage of cells expressing INF2 A149D and INF2 3LA displaying mild or severe nuclear phenotypes. More than 300 cells were examined across three independent experiments. The contrast in the Cherry channel was increased as indicated to identify the cells expressing mutant INF2. Nuclei were visualized with DAPI. Scale bars, 5 μm ( A ) and 10 μm ( B , E ). **, p < 0.01; ***, p < 0.001

Figure Legend Snippet: INF2 R218Q requires integrity of its actin polymerization activity to produce nuclear abnormalities. A Dynamics of wt INF2 during mitosis in MDCK cells expressing GFP-tubulin. Cells were stained with SiR-DNA. B Images of INF2 KO MDCK cells expressing INF2 R218Q and R218Q IA. ( C ) Box plots showing the intensity of the perinuclear F-actin and Cherry corresponding to INF2-1 R218Q and INF2-1 R218Q IA MDCK KO cells. More than 150 cells were examined; three independent experiments. D Percentage of MDCK KO cells expressing INF2-1 R218Q IA and INF2-2 R218Q IA exhibiting mild or severe nuclear phenotypes. More than 350 cells were examined; three independent experiments. E Images of an equatorial plane of INF2 KO MDCK cells expressing INF2 A149D and INF2 3LA. F Percentage of cells expressing INF2 A149D and INF2 3LA displaying mild or severe nuclear phenotypes. More than 300 cells were examined across three independent experiments. The contrast in the Cherry channel was increased as indicated to identify the cells expressing mutant INF2. Nuclei were visualized with DAPI. Scale bars, 5 μm ( A ) and 10 μm ( B , E ). **, p < 0.01; ***, p < 0.001

Techniques Used: Activity Assay, Expressing, Staining, Mutagenesis

Image Search Results

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: INF2 R218Q induces the formation of multipolar spindles in MDCK cells. A Percentage of Cherry and INF2 R218Q cells in mitosis after 48 h of expression. More than 1000 cells were analyzed for each experimental condition in five independent experiments. B Distribution of Cherry and INF2 R218Q cells across different phases of the cell cycle. More than 200 cells were analyzed for each experimental condition, four independent experiments. C Percentage of mitotic Cherry and INF2 R218Q cells displaying multipolar spindles. 68 Cherry cells and 152 INF2 R218Q cells were examined; four independent experiments. D Images of INF2 R218Q mitotic cells stained for centrin, α-tubulin and γ-tubulin. Nuclei were visualized using DAPI. E Classification of INF2 R218Q mitotic cells based on the number of MTOCs expressed as percentage of total cells. MTOCs were identified by γ-tubulin and α-tubulin staining. Over 200 cells were examined; four independent experiments. F Top: Schematic of centriole arrangements. Bottom: centriole arrangement, as visualized with centrin, in INF2 R218Q mitotic cells categorized as clustered in two centrosomes (2 + 2), disengaged in one (2 + 1 + 1) or both (1 + 1 + 1 + 1) centrosomes, or with an abnormal number of centrioles, presented as a percentage of total cells. More than 170 cells were examined; three independent experiments. G Top panel: schematic of ER invasion of the spindle space in cells expressing pathogenic INF2. Bottom panels: INF2 L76P cells stably expressing GFP-sec61 and stained with SiR-DNA analyzed by videomicroscopy during mitosis. The arrowheads indicate regions of the mitotic spindle invaded by ER membranes. H The graph shows the percentage of wt INF2 and INF2 R218Q cells with the spindle space invaded by ER membranes. More than 80 cells were analyzed; three independent experiments. I Videomicroscopic analysis of INF2 R218Q cells stably expressing GFP-sec61 and stained with SiR-DNA during formation of multiple micronuclei. Scale bars, 5 μm. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The DNA constructs expressing GFP fused to centrin-1 (# 72641), centrin-2 (# 41147), and centrin-3 (# 69746), CaM (# 47602), H2B (# 11680), sec61β (# 62008), HEC1 (# 114049) and α-tubulin (# 58197) were obtained from Addgene.

Techniques: Expressing, Staining, Stable Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: INF2 R218Q induces nuclear accumulation of p53 and activation of caspase-3 in MDCK cells. A The graph depicts the time from prophase initiation to the end of cell division, as determined by time-lapse analysis, in control cells (n = 63) and in cells expressing pathogenic INF2 ending with mild (n = 48 cells) and severe (n = 36 cells) nuclear defects; three independent experiments were performed. MDCK cells stably expressing H2B-GFP were used in this experiment. B Immunoblot analysis of p53 in wt INF2 and INF2 R218Q cells. GAPDH was used as loading control. C Quantification of p53 levels in wt INF2 and R218Q cells. Five independent experiments were performed. D Image of p53 staining in an equatorial plane of wt INF2 and INF2 R218 cells. Cells were stained with anti-Cherry antibodies to help visualization of the exogenous proteins. E Percentage of cells with nuclear p53, considering staining in the main nucleus or micronuclei as positive. Over 350 cells were examined; three independent experiments. F Images of cleaved caspase-3 staining in apoptotic INF2 R218Q cells (top) and normal cells (bottom). Nuclei were visualized with DAPI. G Percentage of Cherry, wt INF2 and INF2 R218Q cells positive for cleaved caspase-3 after 48 h of expression. More than 700 cells were examined per experimental condition; three independent experiments. H The percentage of cells with normal, mild or severe nuclear morphology or in mitosis that died between 55 and 72 h of INF2 R218Q expression was determined by time-lapse microscopy in cells labeled with SiR-DNA. 130 cells were analyzed; three independent experiments. I Videomicroscopic analysis of a mitotic (left panels) and an INF2 R218Q cell with a severe phenotype (right panels) undergoing death and detachment from the substrate. Arrowheads indicate the dying cell. Nuclei were stained with SiR-DNA. DIC, differential interference contrast microscopy. Scale bars, 10 μm ( D , F ), 250 μm ( I ). n.s., not significant; **, p < 0.01; ***, p < 0.001

Techniques: Activation Assay, Control, Expressing, Stable Transfection, Western Blot, Staining, Time-lapse Microscopy, Labeling, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: INF2 R218Q requires integrity of its actin polymerization activity to produce nuclear abnormalities. A Dynamics of wt INF2 during mitosis in MDCK cells expressing GFP-tubulin. Cells were stained with SiR-DNA. B Images of INF2 KO MDCK cells expressing INF2 R218Q and R218Q IA. ( C ) Box plots showing the intensity of the perinuclear F-actin and Cherry corresponding to INF2-1 R218Q and INF2-1 R218Q IA MDCK KO cells. More than 150 cells were examined; three independent experiments. D Percentage of MDCK KO cells expressing INF2-1 R218Q IA and INF2-2 R218Q IA exhibiting mild or severe nuclear phenotypes. More than 350 cells were examined; three independent experiments. E Images of an equatorial plane of INF2 KO MDCK cells expressing INF2 A149D and INF2 3LA. F Percentage of cells expressing INF2 A149D and INF2 3LA displaying mild or severe nuclear phenotypes. More than 300 cells were examined across three independent experiments. The contrast in the Cherry channel was increased as indicated to identify the cells expressing mutant INF2. Nuclei were visualized with DAPI. Scale bars, 5 μm ( A ) and 10 μm ( B , E ). **, p < 0.01; ***, p < 0.001

Techniques: Activity Assay, Expressing, Staining, Mutagenesis

TULP3 determines localization of multiple rhodopsin family GPCRs to primary cilia. (A) Stable RPE hTERT or IMCD3 Flp-In cell lines expressing the indicated GPCRs C-terminally tagged with GFP were transfected with 100 nM TULP3 si #3 siRNA (for RPE, see Materials and methods) or were sequentially transfected with 200 nM Tulp3 siRNA twice (for IMCD3 expressing P2RY1, see Materials and methods) for 72 h and serum starved for the last 24 h before fixation and then were immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted from two experiments, and total counted cells are >200 for each condition. Data represent means ± SD. (B) Same as in A, with RPE hTERT stable lines expressing GPR83 and KISS1R, except the ciliary intensity of GFP was quantified. Total counted cells are >100 for each condition. A.U., arbitrary units. (C) Stable IMCD3 Flp-In cells expressing D1R C-terminally tagged with GFP were sequentially transfected with 200 nM Tulp3 siRNA twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and staining for Gpr161, acetylated tubulin, and DNA. D1R GFP /Gpr161-positive cilia were counted from two experiments, and total counted cells are 300–500 for each condition. Data represent means ± SD. (D) IMCD3 Flp-In cells stably expressing LAP TULP3 (LAP; S tag–PreScission-GFP) were sequentially transfected with 200 nM Tulp3 siRNA twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and staining for Gpr161, acetylated tubulin, and DNA. Data represent means ± SD from three experiments, and total counted cells are >400 for each condition. (A–D) *, P < 0.01 (A); *, P < 0.001 (B); *, P < 0.05 (C; A–C, with respect to corresponding siRNA controls); and *, P < 0.001 (D). (E) Table summarizing rhodopsin family GPCRs tested for ciliary localization and the role of TULP3/TUB in trafficking. Unlike the long D2R isoform, the D2R short isoform (NCBI RefSeq database accession no. NP_057658 ) is ciliary . ND, not determined or ciliary, but limited stable expression in RPE hTERT cells. Other class A GPCRs reported to be ciliary by transient expression or endogenously in neurons or thyrocytes but not detected in cilia upon stable expression in RPE hTERT cells include the neuromedin receptor NMUR1 , the orphan PGR15L, the pyroglutamylated RFamide peptide (QRFP) receptor QRFPR , and the trace amine receptor TAAR1 . Two other cilia-localized seven-transmembrane (7TM) receptors, GPR157 and Gpr175 (TPRA1; ), that were not tested are either not readily classifiable or have no homology to known GPCRs, respectively ( ; ; http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=113 ). Also see Fig. S1.

Journal: The Journal of Cell Biology

Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

doi: 10.1083/jcb.201607095

Figure Lengend Snippet: TULP3 determines localization of multiple rhodopsin family GPCRs to primary cilia. (A) Stable RPE hTERT or IMCD3 Flp-In cell lines expressing the indicated GPCRs C-terminally tagged with GFP were transfected with 100 nM TULP3 si #3 siRNA (for RPE, see Materials and methods) or were sequentially transfected with 200 nM Tulp3 siRNA twice (for IMCD3 expressing P2RY1, see Materials and methods) for 72 h and serum starved for the last 24 h before fixation and then were immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted from two experiments, and total counted cells are >200 for each condition. Data represent means ± SD. (B) Same as in A, with RPE hTERT stable lines expressing GPR83 and KISS1R, except the ciliary intensity of GFP was quantified. Total counted cells are >100 for each condition. A.U., arbitrary units. (C) Stable IMCD3 Flp-In cells expressing D1R C-terminally tagged with GFP were sequentially transfected with 200 nM Tulp3 siRNA twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and staining for Gpr161, acetylated tubulin, and DNA. D1R GFP /Gpr161-positive cilia were counted from two experiments, and total counted cells are 300–500 for each condition. Data represent means ± SD. (D) IMCD3 Flp-In cells stably expressing LAP TULP3 (LAP; S tag–PreScission-GFP) were sequentially transfected with 200 nM Tulp3 siRNA twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and staining for Gpr161, acetylated tubulin, and DNA. Data represent means ± SD from three experiments, and total counted cells are >400 for each condition. (A–D) *, P < 0.01 (A); *, P < 0.001 (B); *, P < 0.05 (C; A–C, with respect to corresponding siRNA controls); and *, P < 0.001 (D). (E) Table summarizing rhodopsin family GPCRs tested for ciliary localization and the role of TULP3/TUB in trafficking. Unlike the long D2R isoform, the D2R short isoform (NCBI RefSeq database accession no. NP_057658 ) is ciliary . ND, not determined or ciliary, but limited stable expression in RPE hTERT cells. Other class A GPCRs reported to be ciliary by transient expression or endogenously in neurons or thyrocytes but not detected in cilia upon stable expression in RPE hTERT cells include the neuromedin receptor NMUR1 , the orphan PGR15L, the pyroglutamylated RFamide peptide (QRFP) receptor QRFPR , and the trace amine receptor TAAR1 . Two other cilia-localized seven-transmembrane (7TM) receptors, GPR157 and Gpr175 (TPRA1; ), that were not tested are either not readily classifiable or have no homology to known GPCRs, respectively ( ; ; http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=113 ). Also see Fig. S1.

Article Snippet: Cells were transfected with DNA constructs expressing GFP TULP3 fragments under the chicken β-actin promoter after 5 d in culture using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Transfection, Staining, Stable Transfection

CLSs from GPCRs and fibrocystin require Tulp3 for trafficking to cilia. (A) CLS wild-type and mutant sequences used for making CD8-CLS-LAP and CLS-LAP fusions (left), and a diagram representing the CD8-CLS-LAP or CLS-LAP fusions (right). EC, extracellular region; IC, intracellular region; TM, transmembrane domain. (B) Stable RPE lines expressing the indicated CD8-CLS-LAP or CLS-LAP fusions were transfected with 100 nM TULP3 siRNA for 72 h, serum starved for the last 24 h, and processed as in . GFP-positive cilia were counted in three experiments, and total counted cells are >300 in each condition. Data represent means ± SD; *, P < 0.001 with respect to corresponding siRNA controls. Also see Fig. S2.

Journal: The Journal of Cell Biology

Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

doi: 10.1083/jcb.201607095

Figure Lengend Snippet: CLSs from GPCRs and fibrocystin require Tulp3 for trafficking to cilia. (A) CLS wild-type and mutant sequences used for making CD8-CLS-LAP and CLS-LAP fusions (left), and a diagram representing the CD8-CLS-LAP or CLS-LAP fusions (right). EC, extracellular region; IC, intracellular region; TM, transmembrane domain. (B) Stable RPE lines expressing the indicated CD8-CLS-LAP or CLS-LAP fusions were transfected with 100 nM TULP3 siRNA for 72 h, serum starved for the last 24 h, and processed as in . GFP-positive cilia were counted in three experiments, and total counted cells are >300 in each condition. Data represent means ± SD; *, P < 0.001 with respect to corresponding siRNA controls. Also see Fig. S2.

Article Snippet: Cells were transfected with DNA constructs expressing GFP TULP3 fragments under the chicken β-actin promoter after 5 d in culture using Lipofectamine 2000 (Invitrogen).

Techniques: Mutagenesis, Expressing, Transfection

Proximity biotinylation assays determine CLS–Tulp3 membrane proximity. (A) T-Rex-293 cells were cotransfected with CD8-CLS-BirA* and LAP-TULP3 constructs and processed for a tandem immunopurification (IP) procedure for detecting and quantifying the extent of biotin labeling on LAP-TULP3 as indicated (left). Diagram representing the fusion constructs appears on the right. BirA* will biotinylate primary amine-containing lysine residues in TULP3 fusion, if in close proximity. (B) Western blots of inputs and tandem affinity IPs for the indicated conditions are shown after pretreatment with biotin (50 µM) for 24 h. IB for biotin refers to neutravidin-tagged IRDye 680RD antibody-based detection. Normalized (Norm.) IP values refer to biotin/S tag signal in corresponding S tag secondary IPs, with respect to the control linker biotin-pretreated sample. The number of experiments (N) performed is mentioned beneath each panel. Normalized IP values are reported as means ± SD. (C) T-Rex-293 cells were cotransfected with CD8-CLS-BirA* and the LAP-TULP3 wild type (WT) or PI(4,5)P 2 binding-deficient mutant (TULP3 K268A;R270A ), treated with biotin (20 µM) for 12 h, and processed for tandem IP procedure for detecting and quantifying the extent of biotin labeling as in A. Normalized IP values are reported as means ± SD from two experiments; **, P < 0.01; ***, P < 0.001, with respect to respective control linker biotin-pretreated samples. Also see Figs. S3 and S4.

Journal: The Journal of Cell Biology

Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

doi: 10.1083/jcb.201607095

Figure Lengend Snippet: Proximity biotinylation assays determine CLS–Tulp3 membrane proximity. (A) T-Rex-293 cells were cotransfected with CD8-CLS-BirA* and LAP-TULP3 constructs and processed for a tandem immunopurification (IP) procedure for detecting and quantifying the extent of biotin labeling on LAP-TULP3 as indicated (left). Diagram representing the fusion constructs appears on the right. BirA* will biotinylate primary amine-containing lysine residues in TULP3 fusion, if in close proximity. (B) Western blots of inputs and tandem affinity IPs for the indicated conditions are shown after pretreatment with biotin (50 µM) for 24 h. IB for biotin refers to neutravidin-tagged IRDye 680RD antibody-based detection. Normalized (Norm.) IP values refer to biotin/S tag signal in corresponding S tag secondary IPs, with respect to the control linker biotin-pretreated sample. The number of experiments (N) performed is mentioned beneath each panel. Normalized IP values are reported as means ± SD. (C) T-Rex-293 cells were cotransfected with CD8-CLS-BirA* and the LAP-TULP3 wild type (WT) or PI(4,5)P 2 binding-deficient mutant (TULP3 K268A;R270A ), treated with biotin (20 µM) for 12 h, and processed for tandem IP procedure for detecting and quantifying the extent of biotin labeling as in A. Normalized IP values are reported as means ± SD from two experiments; **, P < 0.01; ***, P < 0.001, with respect to respective control linker biotin-pretreated samples. Also see Figs. S3 and S4.

Article Snippet: Cells were transfected with DNA constructs expressing GFP TULP3 fragments under the chicken β-actin promoter after 5 d in culture using Lipofectamine 2000 (Invitrogen).

Techniques: Membrane, Construct, Immu-Puri, Labeling, Western Blot, Control, Binding Assay, Mutagenesis

CLSs and TULP3 are chemically cross-linked in a ciliary sequence-specific manner. (A–C) T-Rex-293 cells were cotransfected with CD8-CLS-LAP and 6×Myc-TULP3 full-length (B) or 6×Myc–C-terminal tubby domain (C term, 184–442 aa; C) constructs, and processed for cross-linking, quenching, tandem IP procedure, and decross-linking as indicated in the format in the left panel in A. The right panel in A shows a diagram representing the fusion constructs. Western blots of inputs and tandem affinity IPs for the indicated conditions are shown in B and C. Cross-linking enrichment scores shown beneath each respective panel represents (Myc IP/S tag IP) DSP – (Myc IP/S tag IP) NoDSP for each CD8-CLS-LAP–transfected condition normalized with respect to control linker (B) and wild-type CLSs in individual panels in C. The number of lysines in the linker/CLS are as follows: linker GSGAAAAAGSG (0); β 2 andrenergic receptor IC3 (β 2 AR IC3; RVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKT; 5); Fibro WT CLS (12); Fibro IKP CLS (11); Fibro 7A CLS (9); Gpr161 WT IC3 (4); Gpr161 5A IC3 (2); Gpr161 3A IC3 (2); Mchr1 WT IC3 (1); and Mchr1 5A IC3 (1), as shown in . In addition, the rest of the fusion in CD8-CLS-LAP, including CD8α, the attB recombinase site, S tag, and the PreScission protease site from LAP, has a total of 11 lysines. All experiments were performed twice, except the last panel in C, which was performed three times. (B and C) Cross-linking enrichment scores are reported as means ± SD; *, P < 0.5 with respect to control linker (B); *, P < 0.05; **, P < 0.01; ***, P < 0.001with respect to wild-type CLS in each panel (C).

Journal: The Journal of Cell Biology

Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

doi: 10.1083/jcb.201607095

Figure Lengend Snippet: CLSs and TULP3 are chemically cross-linked in a ciliary sequence-specific manner. (A–C) T-Rex-293 cells were cotransfected with CD8-CLS-LAP and 6×Myc-TULP3 full-length (B) or 6×Myc–C-terminal tubby domain (C term, 184–442 aa; C) constructs, and processed for cross-linking, quenching, tandem IP procedure, and decross-linking as indicated in the format in the left panel in A. The right panel in A shows a diagram representing the fusion constructs. Western blots of inputs and tandem affinity IPs for the indicated conditions are shown in B and C. Cross-linking enrichment scores shown beneath each respective panel represents (Myc IP/S tag IP) DSP – (Myc IP/S tag IP) NoDSP for each CD8-CLS-LAP–transfected condition normalized with respect to control linker (B) and wild-type CLSs in individual panels in C. The number of lysines in the linker/CLS are as follows: linker GSGAAAAAGSG (0); β 2 andrenergic receptor IC3 (β 2 AR IC3; RVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKT; 5); Fibro WT CLS (12); Fibro IKP CLS (11); Fibro 7A CLS (9); Gpr161 WT IC3 (4); Gpr161 5A IC3 (2); Gpr161 3A IC3 (2); Mchr1 WT IC3 (1); and Mchr1 5A IC3 (1), as shown in . In addition, the rest of the fusion in CD8-CLS-LAP, including CD8α, the attB recombinase site, S tag, and the PreScission protease site from LAP, has a total of 11 lysines. All experiments were performed twice, except the last panel in C, which was performed three times. (B and C) Cross-linking enrichment scores are reported as means ± SD; *, P < 0.5 with respect to control linker (B); *, P < 0.05; **, P < 0.01; ***, P < 0.001with respect to wild-type CLS in each panel (C).

Article Snippet: Cells were transfected with DNA constructs expressing GFP TULP3 fragments under the chicken β-actin promoter after 5 d in culture using Lipofectamine 2000 (Invitrogen).

Techniques: Sequencing, Construct, Western Blot, Transfection, Control

Differential effects of Tub on ciliary GPCR trafficking. (A) IMCD3 Flp-In cells stably expressing LAP–TUB isoform b were sequentially transfected with 200 nM of the indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted in two experiments, and total counted cells are >400/condition. Data represent means ± SD. Bars, 5 µm. (B) MBP TUB isoform b and MBP TULP3 immobilized on amylose resin were incubated with PreScission eluates from IFT140 LAP RPE cells ± His TULP3 (1–183 aa; see Materials and methods; ). LAP, S tag–PreScission-GFP. MBP TUB- and MBP TULP3-bound proteins and corresponding flowthroughs were immunoblotted for S tag (IFT140 S tag ), maltose-binding protein (MBP), and His-tag as indicated. Data are representative of two experiments. (C and D) Embryonic day 16.5, day in vitro (DIV) 8 hippocampal neurons from wild-type (WT) and Tub mice were immunostained for Gpr161/Gpr19 (green), DyLight594-labeled ACIII (red), and Sstr3 (white; C). Gpr161/Gpr19-positive and -negative cilia are marked by arrows (A and C) and arrowheads (C), respectively. Gpr161/Sstr3 coexpressing cells are marked by yellow arrows. The red dot indicates debris under the coverslip. Data represents means ± SD from cultures of two different embryos belonging to each genotype. Total counted cells are >300 per condition for Gpr161/Sstr3 and >90 per condition for Gpr19. Bars, 5 µm. Ciliary lengths are 3.2 ± 0.1 µm (wild type) and 3.5 ± 0.1 µm ( Tub ) neurons in experiments with Gpr161 staining and 3.7 ± 0.9 µm (wild type) and 3.5 ± 0.8 µm ( Tub ) in experiments with Gpr19 staining. Data represent means ± SD. n > 50 each. (E) Embryonic day 16.5 DIV5 hippocampal neurons from wild-type mice were transfected with indicated GFP-tagged N terminus (NT) wild-type or non–IFT-A binding ( mut12 ) TULP3 constructs , fixed at DIV8, and immunostained for Gpr161 and DyLight594-labeled ACIII. GFP-positive cells were quantified for Gpr161-positive cilia from two different coverslips, and total counted neurons are >24 per condition. (F) Embryonic day 18.5 DIV5 glia from wild-type mice were transfected with constructs as in C, fixed at DIV8, and immunostained for Gpr19 and Arl13b. GFP-positive cells were quantified for Gpr19-positive cilia from cultures from two different mice, and total counted cells are >110 per condition. (A and D–F) *, P < 0.001 with respect to corresponding siRNA control (A); *, P < 0.05 with respect to corresponding wild type; ns, not significant (D); *, P < 0.05 with respect to GFP TULP3-NTmut12 transfected cells (E); and *, P < 0.05 with respect to GFP TULP3-NTmut12 -transfected cells. Also see Fig. S4.

Journal: The Journal of Cell Biology

Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

doi: 10.1083/jcb.201607095

Figure Lengend Snippet: Differential effects of Tub on ciliary GPCR trafficking. (A) IMCD3 Flp-In cells stably expressing LAP–TUB isoform b were sequentially transfected with 200 nM of the indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted in two experiments, and total counted cells are >400/condition. Data represent means ± SD. Bars, 5 µm. (B) MBP TUB isoform b and MBP TULP3 immobilized on amylose resin were incubated with PreScission eluates from IFT140 LAP RPE cells ± His TULP3 (1–183 aa; see Materials and methods; ). LAP, S tag–PreScission-GFP. MBP TUB- and MBP TULP3-bound proteins and corresponding flowthroughs were immunoblotted for S tag (IFT140 S tag ), maltose-binding protein (MBP), and His-tag as indicated. Data are representative of two experiments. (C and D) Embryonic day 16.5, day in vitro (DIV) 8 hippocampal neurons from wild-type (WT) and Tub mice were immunostained for Gpr161/Gpr19 (green), DyLight594-labeled ACIII (red), and Sstr3 (white; C). Gpr161/Gpr19-positive and -negative cilia are marked by arrows (A and C) and arrowheads (C), respectively. Gpr161/Sstr3 coexpressing cells are marked by yellow arrows. The red dot indicates debris under the coverslip. Data represents means ± SD from cultures of two different embryos belonging to each genotype. Total counted cells are >300 per condition for Gpr161/Sstr3 and >90 per condition for Gpr19. Bars, 5 µm. Ciliary lengths are 3.2 ± 0.1 µm (wild type) and 3.5 ± 0.1 µm ( Tub ) neurons in experiments with Gpr161 staining and 3.7 ± 0.9 µm (wild type) and 3.5 ± 0.8 µm ( Tub ) in experiments with Gpr19 staining. Data represent means ± SD. n > 50 each. (E) Embryonic day 16.5 DIV5 hippocampal neurons from wild-type mice were transfected with indicated GFP-tagged N terminus (NT) wild-type or non–IFT-A binding ( mut12 ) TULP3 constructs , fixed at DIV8, and immunostained for Gpr161 and DyLight594-labeled ACIII. GFP-positive cells were quantified for Gpr161-positive cilia from two different coverslips, and total counted neurons are >24 per condition. (F) Embryonic day 18.5 DIV5 glia from wild-type mice were transfected with constructs as in C, fixed at DIV8, and immunostained for Gpr19 and Arl13b. GFP-positive cells were quantified for Gpr19-positive cilia from cultures from two different mice, and total counted cells are >110 per condition. (A and D–F) *, P < 0.001 with respect to corresponding siRNA control (A); *, P < 0.05 with respect to corresponding wild type; ns, not significant (D); *, P < 0.05 with respect to GFP TULP3-NTmut12 transfected cells (E); and *, P < 0.05 with respect to GFP TULP3-NTmut12 -transfected cells. Also see Fig. S4.

Article Snippet: Cells were transfected with DNA constructs expressing GFP TULP3 fragments under the chicken β-actin promoter after 5 d in culture using Lipofectamine 2000 (Invitrogen).

Techniques: Stable Transfection, Expressing, Transfection, Incubation, Binding Assay, In Vitro, Labeling, Staining, Construct, Control

PC1/2 trafficking to cilia requires Tulp3. (A) mIMCD-K2 cells were sequentially transfected with the indicated 200 nM siRNAs twice for 72 h and serum starved for the last 36 h before fixation and immunostained for PC2 using anti-mouse PC2 rabbit polyclonal serum (from G. Pazour; see the Antibodies section of Materials and methods), acetylated tubulin, and DNA. PC2-positive and -negative cilia are marked by white arrows and arrowheads, respectively. The inset shows PC2 levels in control and Tulp3 siRNA-treated cells by immunoblotting. Data represent means ± SD from three experiments. The total number of cells counted is >800 per condition. Quantification of PC2 using a separate antibody available commercially in Fig. S5 (A and B). (B) mIMCD-K2 cells expressing LAP TULP3 were treated as in A. The total counted cells are >350 for each condition. ns, not significant. (C) mIMCD-K2 cells stably expressing Flag PC1 HA were sequentially transfected with the indicated 200-nM siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 36 h before fixation and immunostained for Flag (green), HA (red), AcTub (magenta), and DNA. N-terminal Flag tag allowed determination of PC1-expressing cells. Flag-positive cells were counted for HA-positive cilia from three independent experiments, and total counted cells were >1,000 per condition with ∼10% cells being Flag positive. PC1 HA -positive and -negative cilia are marked by white arrows and arrowheads, respectively. The magenta arrow marks a cilium in a cell not expressing Flag. Data represent means ± SD. (D) ARPE cells stably expressing PC2 L703X GFP were transfected with the indicated 100 nM siRNAs for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, pericentrin, and DNA. GFP-positive cilia were counted from two experiments, and the total number of GFP-positive cells counted were >120 per condition. GFP-positive and -negative cilia are marked by white arrows and arrowheads, respectively. Yellow arrows point to perinuclear staining for PC2 L703X GFP. Data represent means ± SD. Ciliary lengths of PC2 L703X GFP-positive cells treated with control and TULP3 siRNA are 8.5 ± 4.6 and 3.2 ± 0.8 µm, respectively. n = 20 each. (E) mIMCD-K2 cells were transfected with indicated siRNAs as in A and immunostained for PC2 (using anti-mouse PC2 rabbit polyclonal serum) or Gpr161, acetylated tubulin, and DNA. Pixel intensities of PC2- and Gpr161-positive cilia are shown to the right. White arrows mark PC2-positive cilia, and yellow arrows point to cilia shown in insets. Total counted cells were >240 and >140 per condition for PC2 and Gpr161 immunofluorescence, respectively. (A and C–E) *, P < 0.001 with respect to corresponding siRNA control (A); *, P < 0.01 with respect to corresponding siRNA control (C and D); *, P < 0.0001 with respect to controls (E). A.U., arbitrary units. Bars: (A and C–E, main images) 5 µm; (E, insets) 1 µm. Also see Fig. S5.

Journal: The Journal of Cell Biology

Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

doi: 10.1083/jcb.201607095

Figure Lengend Snippet: PC1/2 trafficking to cilia requires Tulp3. (A) mIMCD-K2 cells were sequentially transfected with the indicated 200 nM siRNAs twice for 72 h and serum starved for the last 36 h before fixation and immunostained for PC2 using anti-mouse PC2 rabbit polyclonal serum (from G. Pazour; see the Antibodies section of Materials and methods), acetylated tubulin, and DNA. PC2-positive and -negative cilia are marked by white arrows and arrowheads, respectively. The inset shows PC2 levels in control and Tulp3 siRNA-treated cells by immunoblotting. Data represent means ± SD from three experiments. The total number of cells counted is >800 per condition. Quantification of PC2 using a separate antibody available commercially in Fig. S5 (A and B). (B) mIMCD-K2 cells expressing LAP TULP3 were treated as in A. The total counted cells are >350 for each condition. ns, not significant. (C) mIMCD-K2 cells stably expressing Flag PC1 HA were sequentially transfected with the indicated 200-nM siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 36 h before fixation and immunostained for Flag (green), HA (red), AcTub (magenta), and DNA. N-terminal Flag tag allowed determination of PC1-expressing cells. Flag-positive cells were counted for HA-positive cilia from three independent experiments, and total counted cells were >1,000 per condition with ∼10% cells being Flag positive. PC1 HA -positive and -negative cilia are marked by white arrows and arrowheads, respectively. The magenta arrow marks a cilium in a cell not expressing Flag. Data represent means ± SD. (D) ARPE cells stably expressing PC2 L703X GFP were transfected with the indicated 100 nM siRNAs for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, pericentrin, and DNA. GFP-positive cilia were counted from two experiments, and the total number of GFP-positive cells counted were >120 per condition. GFP-positive and -negative cilia are marked by white arrows and arrowheads, respectively. Yellow arrows point to perinuclear staining for PC2 L703X GFP. Data represent means ± SD. Ciliary lengths of PC2 L703X GFP-positive cells treated with control and TULP3 siRNA are 8.5 ± 4.6 and 3.2 ± 0.8 µm, respectively. n = 20 each. (E) mIMCD-K2 cells were transfected with indicated siRNAs as in A and immunostained for PC2 (using anti-mouse PC2 rabbit polyclonal serum) or Gpr161, acetylated tubulin, and DNA. Pixel intensities of PC2- and Gpr161-positive cilia are shown to the right. White arrows mark PC2-positive cilia, and yellow arrows point to cilia shown in insets. Total counted cells were >240 and >140 per condition for PC2 and Gpr161 immunofluorescence, respectively. (A and C–E) *, P < 0.001 with respect to corresponding siRNA control (A); *, P < 0.01 with respect to corresponding siRNA control (C and D); *, P < 0.0001 with respect to controls (E). A.U., arbitrary units. Bars: (A and C–E, main images) 5 µm; (E, insets) 1 µm. Also see Fig. S5.

Article Snippet: Cells were transfected with DNA constructs expressing GFP TULP3 fragments under the chicken β-actin promoter after 5 d in culture using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Control, Western Blot, Expressing, Stable Transfection, FLAG-tag, Staining, Immunofluorescence

TULP3/TUB-mediated trafficking to ciliary membrane. (A) IMCD3 cells stably expressing SNAP Gpr161 GFP were sequentially transfected with 200 nM of the indicated siRNAs twice as indicated in the experimental format on the top left (see Materials and methods). Starved cells were blocked with SNAP–surface block for 30 min, washed, and then were treated with SNAP–surface 594 at indicated time points. Cells were fixed and immunostained for GFP (green) and acetylated tubulin (magenta). Cilia positive and negative for SNAP–surface 594 are marked by white arrows and arrowheads, respectively. The yellow arrowhead points to cilia faintly stained for SNAP–surface 594. Total counted cells are >150 for control and >200 for Tulp3 siRNA, respectively. Data represent means ± SD from two or more fields from a single experiment. Bars, 5 µm. (B) NIH 3T3 Flp-In cells stably expressing Gpr161-GFP or Gpr161 V158E -GFP mutants were sequentially transfected with 200 nM of indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and immunostaining for GFP, acetylated tubulin, and DNA. Ciliary pixel intensities for GFP are shown. Total counted cells were >60 per condition from two independent transfections with >30 cells counted per coverslip. A.U., arbitrary units. Also see Fig. S5 E. (C) IMCD3 Flp-In cells stably and inducibly expressing Myc TULP3 N terminus (1–183 aa) were starved in the presence of doxycycline (Dox) for 20 h (4 µg/ml). After washing, cells were treated ± SAG (500 nM) for indicated time points in starvation medium before fixing and immunostaining for Gpr161, Myc, acetylated tubulin, and DNA. Myc-positive and -negative cells were scored for Gpr161-positive cilia. Total counted cells are >55 and >180 for Myc-positive and -negative cells, respectively, for each time point. Data represent means ± SD from three coverslips. Also see Fig. S5 F. (A–C) *, P < 0.001 with respect to corresponding siRNA control at each time point measured (A); *, P < 0.0001 with respect to corresponding controls (B); *, P < 0.05; **, P < 0.01 with respect to corresponding 0 h time point (C). (D) Model for TULP3/TUB-mediated trafficking to ciliary membrane. See Discussion. BB, basal body; FP, fusion protein; NT, TULP3/TUB IFT-A binding N-terminus domain. Also see Fig. S5.

Journal: The Journal of Cell Biology

Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

doi: 10.1083/jcb.201607095

Figure Lengend Snippet: TULP3/TUB-mediated trafficking to ciliary membrane. (A) IMCD3 cells stably expressing SNAP Gpr161 GFP were sequentially transfected with 200 nM of the indicated siRNAs twice as indicated in the experimental format on the top left (see Materials and methods). Starved cells were blocked with SNAP–surface block for 30 min, washed, and then were treated with SNAP–surface 594 at indicated time points. Cells were fixed and immunostained for GFP (green) and acetylated tubulin (magenta). Cilia positive and negative for SNAP–surface 594 are marked by white arrows and arrowheads, respectively. The yellow arrowhead points to cilia faintly stained for SNAP–surface 594. Total counted cells are >150 for control and >200 for Tulp3 siRNA, respectively. Data represent means ± SD from two or more fields from a single experiment. Bars, 5 µm. (B) NIH 3T3 Flp-In cells stably expressing Gpr161-GFP or Gpr161 V158E -GFP mutants were sequentially transfected with 200 nM of indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and immunostaining for GFP, acetylated tubulin, and DNA. Ciliary pixel intensities for GFP are shown. Total counted cells were >60 per condition from two independent transfections with >30 cells counted per coverslip. A.U., arbitrary units. Also see Fig. S5 E. (C) IMCD3 Flp-In cells stably and inducibly expressing Myc TULP3 N terminus (1–183 aa) were starved in the presence of doxycycline (Dox) for 20 h (4 µg/ml). After washing, cells were treated ± SAG (500 nM) for indicated time points in starvation medium before fixing and immunostaining for Gpr161, Myc, acetylated tubulin, and DNA. Myc-positive and -negative cells were scored for Gpr161-positive cilia. Total counted cells are >55 and >180 for Myc-positive and -negative cells, respectively, for each time point. Data represent means ± SD from three coverslips. Also see Fig. S5 F. (A–C) *, P < 0.001 with respect to corresponding siRNA control at each time point measured (A); *, P < 0.0001 with respect to corresponding controls (B); *, P < 0.05; **, P < 0.01 with respect to corresponding 0 h time point (C). (D) Model for TULP3/TUB-mediated trafficking to ciliary membrane. See Discussion. BB, basal body; FP, fusion protein; NT, TULP3/TUB IFT-A binding N-terminus domain. Also see Fig. S5.

Article Snippet: Cells were transfected with DNA constructs expressing GFP TULP3 fragments under the chicken β-actin promoter after 5 d in culture using Lipofectamine 2000 (Invitrogen).

Techniques: Membrane, Stable Transfection, Expressing, Transfection, Blocking Assay, Staining, Control, Immunostaining, Binding Assay

Review

Structured Review

Images

1) Product Images from "INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death"

Similar Products

Image Search Results