Journal: Neural Regeneration Research

Article Title: AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice

doi: 10.4103/NRR.NRR-D-23-01277

Figure Lengend Snippet: Antibodies and detailed information

Article Snippet: GFP , Mouse , 1:200 , 66002-1-Ig , AB_11182611 , Proteintech , .

Techniques:

Journal: Neural Regeneration Research

Article Title: Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures

doi: 10.4103/1673-5374.391302

Figure Lengend Snippet: Most Atpα -CMT2 flies have motor and sensory defects and an abnormal circadian neuron dendritic morphology. (A) Images of control, Atpα -CMT2, and mutant flies expressing nSybGFP in the neuromuscular junction of third instar larvae. Staining with anti-GFP antibody revealed the morphology of neuromuscular junctions. All Atpα -CMT2 and mutant flies showed a decrease in synapse number and length. We utilized an online post hoc power calculator (https://clincalc.com/stats/Power.aspx) to assess statistical power, with a false positive rate set at 0.05. Except for the statistical power of Atpα mut/+ in the synapse length group, which is 56.4%, the statistical power of the other significantly different groups exceed 83.5%. (B) Plots of synapse number (left) and length (right) in control, Atpα -CMT2, and mutant flies ( n = 6–10). One-way analysis of variance with Tukey’s multiple comparison test were used. (C) Images of control, Atpα -CMT2, and mutant brains with Class IV multidendritic sensory neurons in the third instar larval body wall visualized by ppk -GAL4-driven mCD8-GFP. Except for Atpα D580F/+ and Atpα mut/+ , all other heterozygous Atpα -CMT2 flies showed significantly reduced dendritic branching. (D) Plots of dendritic branch number in control, Atpα -CMT2, and mutant flies ( n = 15–20). Dendritic branch number was compared using the nonparametric Kruskal-Wallis test with Dunn’s multiple comparison test. The statistical powers of the significantly different groups are between 94.7% and 100%. (E) Left: Image of a circadian circuit in a control brain, visualized by staining with anti-PDF antibodies. sLNvs and lLNvs indicate small and large ventral lateral neurons. Right: The region analyzed. (F) Images of representative PDF dorsal axonal projections from control, Atpα -CMT2, and mutant flies taken during the early day (ZT2) and early night (ZT14). Apart from Atpα mut/+ , there was a significant decrease in axonal arbor complexity of the PDF circuit in Atpα -CMT2 flies. (G) Quantification of the total number of intersections between concentric rings and axonal projections at ZT2 and ZT14 ( n = 8–11). Data were analyzed by one-way analysis of variance with Tukey’s multiple comparison test. The statistical powers of the significantly different groups are between 76.9% and 100%. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars represent SEM. Atpα : Na pump α subunit; CMT2: Charcot-Marie-Tooth disease type 2; Gal4: galactose-responsive transcription factor GAL4; GFP: green fluorescent protein; lLNv: large ventral lateral clock neurons; ns: not significant; PDF: pigment-dispersing factor; ppk: polyphosphate kinase; sLNv: small ventral lateral clock neurons; ZT: zeitgeber time.

Article Snippet: To visualize neuromuscular junctions in third instar larvae, D42 -Gal4,UAS-nSyb-GFP flies (Bloomington Drosophila Stock Center, Bloomington, IN, USA, Cat# 9263) were prepared and stained overnight at 4°C with a primary antibody against green fluorescent protein (GFP; 1:1000, Thermo Fisher Scientific, Waltham, MA, USA, Cat# A11120, RRID: AB_221568).

Techniques: Control, Mutagenesis, Expressing, Staining, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CCDC146 is required for sperm flagellum biogenesis and male fertility in mice

doi: 10.1007/s00018-023-05025-x

Figure Lengend Snippet: The knockout of Ccdc146 leads to male infertility. A CCDC146 was related to CCDC38 predicted by the STRING database. B CCDC146 interacted with CCDC38. pCSII-MYC-Ccdc146 was transfected into HEK293T cells with pEGFP-C1-Ccdc38, 48 hours after transfection, cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. C CCDC146 co-localized with CCDC38 and γ-TUBULIN in Hela cells. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 were co-transfected into Hela cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-γ-TUBULIN or anti-GFP antibodies, and the nucleus was stained with DAPI. Scale bar: 5 μm. D The expression of Ccdc146 in different tissues of adult mice. GAPDH was used as a loading control. E The expression of Ccdc146 on different days. TUBULIN was used as a loading control. F The schematic illustration of the knockout strategy for generating Ccdc146−/− mice lacking exon 3–7. Primer-F1, Primer-R1, and Primer-R2 were used as the genotyping primers. G Genotyping of Ccdc146−/− mice. H Western blotting of CCDC146 indicated the depletion efficiency in Ccdc146−/− male mice. I Ccdc146−/− male mice were infertile. The Y axis represents the average number of offspring per litter. Data are presented as means ± SEM. two-tailed Student’s t test; ***P < 0·001. J The number of pups per litter of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. K The sex ratio of the pups of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. L The size of the Ccdc146+/+ and Ccdc146−/− mice testes were unaffected. M Testis from Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference in weight (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. N The body weight of Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. O The testis/body weight ratios in Ccdc146+/+ and Ccdc146−/− male mice were consistent (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns no significance

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and western blotting (WB): mouse anti-GFP (M20004, Abmart; 1:1000 for WB), rabbit anti-MYC (BE2011, EASYBIO; 1:1000 for WB), anti-ODF2 (12058-1-AP, Proteintech; 1:500 for WB, 1:200 for IF), anti-ODF1 (24736-1-AP, Proteintech; 1:1000 for WB), mouse anti-α-Tubulin antibody (AC012, ABclonal; 1:1000 for WB, 1:100 for IF), rabbit anti-β-Tubulin antibody (10068-1-AP, Proteintech; 1:1000 for WB), mouse anti-α/β-Tubulin antibody (ab44928, Abcam; 1:100 for IF), mouse anti-GAPDH antibody (AC002, ABclonal; 1:10,000 for WB), mouse anti-Ac-Tubulin antibody (T7451, Sigma-Aldrich; 1:200 for IF), mouse anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), rabbit anti-DYNLL2 antibody (16811-1-AP, Proteintech; 1:1000 for WB), rabbit anti-TEKT2 antibody (13518-1-AP, Proteintech; 1:1000 for WB), rabbit anti-SPACA9 antibody (26034-1-AP, Proteintech; 1:1000 for WB), rabbit anti-PPIL6 antibody (17452-1-AP, Proteintech; 1:1000 for WB), rabbit anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), mouse anti-SPAG6 antibody (H00008382-M04, Abnova; 1:1000 for WB), mouse anti-SPAG16 antibody (H00079582-M01, Abnova; 1:1000 for WB), rabbit anti-CCDC38 (generated by Dia-an Biotechnology, Wuhan, China; 1:500 for WB), rabbit anti-CCDC146 (generated by Dia-an Biotechnology, Wuhan, China; 1:200 for WB, 1:50 for IF).

Techniques: Knock-Out, Transfection, Immunoprecipitation, Staining, Expressing, Western Blot, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CCDC146 is required for sperm flagellum biogenesis and male fertility in mice

doi: 10.1007/s00018-023-05025-x

Figure Lengend Snippet: CCDC146 may facilitate ODF2 transportation by interacting with CCDC42 and CCDC38. A-C CCDC146 co-localized with γ-TUBULIN, CCDC38, and CCDC42 in NIH3T3 cells before serum starvation. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 or pEGFP-C1-Ccdc42 were co-transfected into NIH3T3 cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-GFP antibodies, and the nucleus was stained with DAPI. D CCDC42 cannot co-localize with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pEGFP-C1-Ccdc42 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-GFP and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. E CCDC38 co-localized with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pEGFP-C1-Ccdc38 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-GFP and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. F CCDC146 cannot co-localize with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pCSII-MYC-Ccdc146 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-MYC and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. G The immunofluorescence of CCDC146 in Ccdc146+/+ and Ccdc146−/− mice. Testis germ cells were stained with anti-CCDC146 antibody, and the nucleus was stained with DAPI. H The immunofluorescence of CCDC146 in Ccdc146+/+ and Ccdc146−/− mice. Spermatozoa from cauda of the epididymis were stained with anti-CCDC146 antibody, and the nucleus was stained with DAPI. Scale bars: 2·5 μm (A-F); 5 μm (G); 10 μm (A-F, H). I CCDC146 may interact with microtubule proteins and microtubule inner proteins. Co-IP of sperm flagellum proteins with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-α-TUBULIN, anti-β-TUBULIN, anti-TEKT2, anti-SPACA9, anti-DYNLL2, anti-PPIL6, anti-NME5, anti-SPAG6, anti-SPAG16, and anti-FLAG (CCDC146) antibodies. MT microtubule; MIP microtubule inner protein; RS radial spoke; CPA central-pair apparatus

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and western blotting (WB): mouse anti-GFP (M20004, Abmart; 1:1000 for WB), rabbit anti-MYC (BE2011, EASYBIO; 1:1000 for WB), anti-ODF2 (12058-1-AP, Proteintech; 1:500 for WB, 1:200 for IF), anti-ODF1 (24736-1-AP, Proteintech; 1:1000 for WB), mouse anti-α-Tubulin antibody (AC012, ABclonal; 1:1000 for WB, 1:100 for IF), rabbit anti-β-Tubulin antibody (10068-1-AP, Proteintech; 1:1000 for WB), mouse anti-α/β-Tubulin antibody (ab44928, Abcam; 1:100 for IF), mouse anti-GAPDH antibody (AC002, ABclonal; 1:10,000 for WB), mouse anti-Ac-Tubulin antibody (T7451, Sigma-Aldrich; 1:200 for IF), mouse anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), rabbit anti-DYNLL2 antibody (16811-1-AP, Proteintech; 1:1000 for WB), rabbit anti-TEKT2 antibody (13518-1-AP, Proteintech; 1:1000 for WB), rabbit anti-SPACA9 antibody (26034-1-AP, Proteintech; 1:1000 for WB), rabbit anti-PPIL6 antibody (17452-1-AP, Proteintech; 1:1000 for WB), rabbit anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), mouse anti-SPAG6 antibody (H00008382-M04, Abnova; 1:1000 for WB), mouse anti-SPAG16 antibody (H00079582-M01, Abnova; 1:1000 for WB), rabbit anti-CCDC38 (generated by Dia-an Biotechnology, Wuhan, China; 1:500 for WB), rabbit anti-CCDC146 (generated by Dia-an Biotechnology, Wuhan, China; 1:200 for WB, 1:50 for IF).

Techniques: Transfection, Staining, Immunofluorescence, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CCDC146 is required for sperm flagellum biogenesis and male fertility in mice

doi: 10.1007/s00018-023-05025-x

Figure Lengend Snippet: CCDC146 interacts with CCDC38 and CCDC42. A CCDC146 interacted with CCDC42. pCSII-MYC-Ccdc146 were transfected into HEK293T cells with pEGFP-C1-Ccdc42 48 hours after transfection; cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. B CCDC146 interacted with CCDC38 and CCDC42 in testis. Co-IP of CCDC38 and CCDC42 with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-CCDC38, anti-CCDC42, anti-CCDC146 and anti-FLAG (CCDC146) antibodies. C, D Western blots showing CCDC38, CCDC42, ODF2, IFT88, IFT20, and ODF1 protein levels in lysates from Ccdc146+/+ and Ccdc146−/− mice testes. GAPDH served as a loading control. ODF2, IFT88, and IFT20 protein levels were reduced in Ccdc146−/− testes compared with Ccdc146+/+ testes. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; *P < 0·1; **P < 0·01. E Binding mode of CCDC38 on the CCDC146 predicted by docking. Detailed interaction network between CCDC38 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light red) are displayed as sticks. H-bonds are displayed in dash lines. F Binding mode of CCDC42 on the CCDC146 predicted by docking. Detailed interaction network between CCDC42 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light yellow) are displayed as sticks. H-bonds are displayed in yellow dashed lines. G Alignment of the mode of CCDC38 and CCDC42 on the CCDC146 predicted by docking. H–I Interactions between CCDC38 and WT or D541, or R547 mutants of truncated CCDC146 were detected by Co-IP assays. J–K Co-IP assays detected interactions between CCDC42 and WT or L232, or Y245 mutants of truncated CCDC146

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and western blotting (WB): mouse anti-GFP (M20004, Abmart; 1:1000 for WB), rabbit anti-MYC (BE2011, EASYBIO; 1:1000 for WB), anti-ODF2 (12058-1-AP, Proteintech; 1:500 for WB, 1:200 for IF), anti-ODF1 (24736-1-AP, Proteintech; 1:1000 for WB), mouse anti-α-Tubulin antibody (AC012, ABclonal; 1:1000 for WB, 1:100 for IF), rabbit anti-β-Tubulin antibody (10068-1-AP, Proteintech; 1:1000 for WB), mouse anti-α/β-Tubulin antibody (ab44928, Abcam; 1:100 for IF), mouse anti-GAPDH antibody (AC002, ABclonal; 1:10,000 for WB), mouse anti-Ac-Tubulin antibody (T7451, Sigma-Aldrich; 1:200 for IF), mouse anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), rabbit anti-DYNLL2 antibody (16811-1-AP, Proteintech; 1:1000 for WB), rabbit anti-TEKT2 antibody (13518-1-AP, Proteintech; 1:1000 for WB), rabbit anti-SPACA9 antibody (26034-1-AP, Proteintech; 1:1000 for WB), rabbit anti-PPIL6 antibody (17452-1-AP, Proteintech; 1:1000 for WB), rabbit anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), mouse anti-SPAG6 antibody (H00008382-M04, Abnova; 1:1000 for WB), mouse anti-SPAG16 antibody (H00079582-M01, Abnova; 1:1000 for WB), rabbit anti-CCDC38 (generated by Dia-an Biotechnology, Wuhan, China; 1:500 for WB), rabbit anti-CCDC146 (generated by Dia-an Biotechnology, Wuhan, China; 1:200 for WB, 1:50 for IF).

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Two Tailed Test, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 interacts CaMKIIδ directly and promotes CaMKIIδ activation. A-B MS/MS spectrum of the peptide showing FTDEYQLFEELGK and ESTESSNTTIEDEDVK from CaMKIIδ. Single-letter abbreviations: K, Lys; V, Val; I, Ile; G, Gly; Q, Gln; L, Leu; N, Asn; E, Glu; T, Thr; S, Ser; F, Phe; D, Asp; Y, Tyr. m/z, mass/charge ratio. C The complex of JOSD2 and CaMKIIδ in NRVMs were detected by Coimmunoprecipitation. NRVMs were transfected with Flag-tagged JOSD2. Lysates from cells were immunoprecipitated with an anti-Flag antibody. CaMKIIδ levels were detected by immunoblotting. IgG was used as a control for IP. D In HEK-293 T cells which express a few endogenous CaMKII, Flag-tagged JOSD2 and GFP-tagged CaMKIIδ were transfected. Lysates from cells were immunoprecipitated with anti-Flag antibody. GFP levels were detected by immunoblotting. IgG was used as a control for IP. E Representative western blot for CaMKIIδ and p-CaMKIIδ in NRVMs with gradient levels of JOSD2. F–G JOSD2 was silenced or overexpressed in NRVMs. Representative western blotting for CaMKIIδ and p-CaMKIIδ in NRVMs. GAPDH was used as a loading control. (H-I) Representative western blotting for CaMKIIδ and p-CaMKIIδ in heart tissues from ISO- and MI-induced mice. J–K Confocal images (J)and representative recordings (K) of SR Ca2+ determined by the Fluo-4 AM fluorescence signal in cardiomyocytes transfected with si-JOSD2 or si-NC before ISO stimulation for 24 h. n = 3 cells in each group. [Scale bar, 50 μm]. L–M Confocal images (L) and representative recordings (M) of SR Ca2+ determined by the Fluo-4 AM fluorescence signal in cardiomyocytes transfected with Flag-JOSD2 or empty vector (EV) before ISO stimulation for 24 h. n = 6 cells in each group. [Scale bar, 50 μm]

Article Snippet: Antibodies against Calcium–calmodulin-dependent protein kinase II delta-Specific (CaMKIIδ; Cat# 20667-1-AP), Flag tag (Flag; Cat# 20543-1-AP), GFP tag (GFP; Cat# 66002-1-AP), beta-Myosin heavy chain (β-MyHC; Cat# 22280-1-AP), Collagen type I (COL-1; Cat# 14695-1-AP), transforming growth factor-beta1 (TGFβ-1; Cat# 21898-1-AP), and atrial natriuretic peptide (ANP; Cat# 27426-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Activation Assay, Tandem Mass Spectroscopy, Transfection, Immunoprecipitation, Western Blot, Fluorescence, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 directly removes ubiquitin from CaMKIIδ at residue K227. A HEK-293 T cells were transfected with GFP-CaMKIIδ, Flag-JOSD2, Myc-Ub (WT), Myc-Ub(K48) and Myc-Ub(K63). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting. B A schematic illustration of the construct with mutated ubiquitination site of CaMKIIδ. (C) HEK-293 T cells were transfected with Flag-JOSD2, Myc-Ub, GFP-CaMKIIδ (WT), GFP-CaMKIIδK(K138R), GFP-CaMKIIδ(K227R) and GFP-CaMKIIδ(K268R). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting. D A schematic illustration of the construct with the mutated active site of JOSD2. E HEK-293 T cells were transfected with GFP-CaMKIIδ, Myc-Ub, Flag-JOSD2(WT) and Flag-JOSD2(C24A). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting

Article Snippet: Antibodies against Calcium–calmodulin-dependent protein kinase II delta-Specific (CaMKIIδ; Cat# 20667-1-AP), Flag tag (Flag; Cat# 20543-1-AP), GFP tag (GFP; Cat# 66002-1-AP), beta-Myosin heavy chain (β-MyHC; Cat# 22280-1-AP), Collagen type I (COL-1; Cat# 14695-1-AP), transforming growth factor-beta1 (TGFβ-1; Cat# 21898-1-AP), and atrial natriuretic peptide (ANP; Cat# 27426-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Residue, Transfection, Western Blot, Construct

Journal: The Journal of Cell Biology

Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

doi: 10.1083/jcb.202311137

Figure Lengend Snippet: Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.

Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

Techniques: Labeling, Membrane, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

doi: 10.1083/jcb.202311137

Figure Lengend Snippet: TMEM24, but not other ER/PM tethers, concentrate as cell–cell junctions. HEK293 cells. (A and B) TMEM24 expressed in one cell generates enlarged ER/PM junctions that are not mirrored by eGFP-ESyt2-positive or eGFP-JPH4-positive junctions in directly adjacent cells. (C) The accumulation of TMEM24-mCherry expressed in one cell at a cell-adjacent ER/PM junction is mirrored by the accumulation of TMEM24-GFP expressed in the adjacent cell (see also ). (D) ER/PM junctions induced by mCherry-ESyt2 and GFP-ESyt2-expressed in two adjacent cells respectively, do not mirror one another across the cell–cell interface. (E) mCherry-JPH4 and GFP-JPH4 do not robustly mirror one another across the cell–cell interface although junctions could be found that seemed to be symmetrically opposed. (F) eGFP-Esyt2 colocalizes with TMEM24 at ER/PM junctions, but is excluded from the cell adjacent junction where TMEM24-GFP is selectively enriched. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (G) Kv2.1(S601,607D)-GFP, a Kv2.1 construct that binds constitutively to the ER protien VAP colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (H) STIM1(D76A)-GFP, a STIM1 construct that constitutively binds the PM, colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right.

Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

Techniques: Fluorescence, Construct, Membrane

Journal: The Journal of Cell Biology

Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

doi: 10.1083/jcb.202311137

Figure Lengend Snippet: Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .

Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

Techniques: Affinity Purification, Western Blot, Construct

Journal: The Journal of Cell Biology

Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

doi: 10.1083/jcb.202311137

Figure Lengend Snippet: A TMEM24-4.1-SynCAM1 complex at sites of cell – cell contact. (A) Schematic of the SynCAM 1(363)-GFP construct. (B) Confocal images of SynCAM1(363)-eGFP expressed in HEK293 cells. The upper panel is a slice at mid z level while the lower panel is shown as a basal surface. Representative example of n = 22 cells. (C) Coexpression of TMEM24-mCherry and SynCAM1(363)-eGFP in HEK293 cells. Images are of a mid-level z-slice (above) or a basal surface (below) of the cell. Plasma membrane labeled with CellBrite 650. Representative example of n = 35 cells. (D) . Coexpression of JPH4-mCherry and SynCAM1(363)-eGFP in HEK293 cells. The plasma membrane was labeled with CellBrite 650. Representative example of n = 28 cells (E) Pearson’s correlation R values of comparisons of the SynCAM1(363)-eGFP fluorescence with the fluorescence of either TMEM24-mCherry or mCherry-JPH4 (P = 6.76E-11, TMEM24 n = 24, JPH4 n = 30). (F) Expression of SynCAM1(363)-eGFP, mCherry-4.1G, and TMEM24-Halo labeled with JF646 HaloTag ligand in HEK293 cells. All three proteins colocalize at ER/PM junctions. Representative example of n = 74 cells. ( G ) Model of the TMEM24-4.1-SynCAM 1 complex. Diagonal lines indicate regions of the micrographs occupied by cells. Scale bars = 5 μm.

Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

Techniques: Construct, Membrane, Labeling, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

doi: 10.1083/jcb.202311137

Figure Lengend Snippet: Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.

Article Snippet: Membranes were probed with mouse anti-GFP (G6539; Sigma-Aldrich) then labeled with LiCor IRDye 680LT Goat α-Mouse (926-68020) and LiCor IRDye 680RD Streptavidin (926-68079).

Techniques: Labeling, Membrane, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

doi: 10.1083/jcb.202311137

Figure Lengend Snippet: Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .

Article Snippet: Membranes were probed with mouse anti-GFP (G6539; Sigma-Aldrich) then labeled with LiCor IRDye 680LT Goat α-Mouse (926-68020) and LiCor IRDye 680RD Streptavidin (926-68079).

Techniques: Affinity Purification, Western Blot, Construct