a helenius  (Addgene inc)

Journal: bioRxiv

Article Title: A PKD-caveolin axis drives secretory carrier biogenesis at the TGN

doi: 10.64898/2026.01.13.699385

Figure Lengend Snippet: (A) Knockdown efficiency of PKD2 and PKD3 in HeLa cells at 72 h after siRNA transfection. (B) Knockdown efficiency of CAV1 and CAV2 in HeLa cells at 72 h after siRNA transfection. Multiple bands in the immunoblot with an anti-CAV1 antibody represent CAV1α, CAV1β, and their modified forms. CAV2 was detected as a single major band in control cells, but as doublet bands in CAV1-depleted cells.

Article Snippet: The plasmids encoding dog CAV1 (WT and P132L)-mEGFP were generous gifts from A. Helenius (ETH Zurich, Institute of Biochemistry, Switzerland; Addgene plasmid #27704 and #27708; ).

Techniques: Knockdown, Transfection, Western Blot, Modification, Control

Journal: bioRxiv

Article Title: A PKD-caveolin axis drives secretory carrier biogenesis at the TGN

doi: 10.64898/2026.01.13.699385

Figure Lengend Snippet: (A) Schematic representations of the CAV1-SBP-EGFP construct and its synchronized transport from the ER to the Golgi complex by using the RUSH system. (B) CAV1-SBP-EGFP transport from the ER to the PM via the Golgi complex in HeLa cells. Scale bar, 10 μm.

Article Snippet: The plasmids encoding dog CAV1 (WT and P132L)-mEGFP were generous gifts from A. Helenius (ETH Zurich, Institute of Biochemistry, Switzerland; Addgene plasmid #27704 and #27708; ).

Techniques: Construct

Journal: bioRxiv

Article Title: A PKD-caveolin axis drives secretory carrier biogenesis at the TGN

doi: 10.64898/2026.01.13.699385

Figure Lengend Snippet: (A) Colocalization of CAV1-SBP-EGFP with mKate2-FM4-HA and mKate2-FM4-PAUF. High magnifications of the boxed areas are shown in the insets. (B) mKate2-FM4-HA transport to the PM upon control (Cont), CAV1, CAV2, and CAV1/2 (CAVs) knockdown at 50 min after transport initiation in HA-HeLa cells. (C and D) HA carrier biogenesis upon control, CAV1, CAV2, and CAV1/2 knockdown in HA-HeLa cells. (D) Quantification of the HA carrier biogenesis. The number of HA carriers at 15 min after the temperature shift to 37°C is shown. Data are means ± SEM (n = 20 cells per condition; ****, P < 0.0001; one-way ANOVA multiple comparison test). (E and F) CARTS biogenesis upon control, CAV1, CAV2, and CAV1/2 knockdown in HeLa cells stably expressing mKate2-FM4-PAUF. (F) Quantification of the CARTS biogenesis. The number of CARTS at 15 min after the temperature shift to 37°C is shown. Data are means ± SEM (n = 20 cells per condition; ****, P < 0.0001; unpaired two-tailed Student’s t test). (G-I) SBP-EGFP-CD-M6PR transport from the ER to endosomes via the Golgi complex upon control and CAV1/2 knockdown in HeLa cells. (H and I) Quantification of SBP-EGFP-CD-M6PR transport from the Golgi complex to endosomes. The number (H) and the median size (I) of SBP-EGFP-CD-M6PR–positive puncta (corresponding to clathrin-coated carriers or endosomal membranes) at 30 min after transport initiation is shown. (H) Data are means ± SEM (n = 20 cells per condition; unpaired two-tailed Student’s t test). (I) Boxes delimit the first and third quartiles, and the central line is the median, whereas the cross represents the mean value. The whiskers represent the minimum and maximum values (n = 20 cells per condition; unpaired two-tailed Student’s t test). Scale bars, 10 μm (large panels), 5 μm (insets).

Article Snippet: The plasmids encoding dog CAV1 (WT and P132L)-mEGFP were generous gifts from A. Helenius (ETH Zurich, Institute of Biochemistry, Switzerland; Addgene plasmid #27704 and #27708; ).

Techniques: Control, Knockdown, Comparison, Stable Transfection, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: A PKD-caveolin axis drives secretory carrier biogenesis at the TGN

doi: 10.64898/2026.01.13.699385

Figure Lengend Snippet: (A) Accumulation of mKate2-FM4-HA at the Golgi complex at 50 min after transport initiation in HA-HeLa cells expressing CAV1 P132L-mEGFP. The cells expressing mKate2-FM4-HA alone were observed as a control. Scale bar, 10 μm. A merged image of CAV1 P132L-mEGFP and mKate2-FM4-HA in the boxed areas is shown in the inset. Scale bars, 10 μm (large panels), 5 μm (inset). (B and C) Membrane tubule formation upon expression of GST-PKD2 KD alone (control) and co-expression of WT or P132L CAV1-mEGFP in HeLa cells. High magnifications of the boxed areas are shown in the insets. Scale bars, 10 μm (large panels), 5 μm (insets). (C) Quantification of the GST-PKD2 KD-induced membrane tubule formation. The percentage of cells with GST-PKD2 KD-positive tubules is shown. Data are means ± SEM (n = 3 independent experiments (100–108 cells per condition); **, P = 0.0089; ***, P = 0.0001; one-way ANOVA multiple comparison test). (D) Accumulation of CAV1 WT-mEGFP at bulging TGN domains connected to GST-PKD2 KD-positive tubules in HeLa cells. 3D super-resolution microscopic images were acquired as described in Materials and methods. Maximum intensity merges of z-stack images are shown. High magnifications of the boxed areas are shown in the right of each panels. Arrowheads indicate CAV1 WT-mEGFP–positive bulging TGN domains connected to GST-PKD2 KD-positive tubules. N, nucleus. Scale bars, 10 μm (left panels), 5 μm (right panels). See also Video 2. (E) Interaction of CAV1-mEGFP with GST-PKD2. Lysates of HEK 293T cells coexpressing GST (control) or GST-PKD2 (WT or KD) with CAV1 (WT or P132L: PL)-mEGFP were incubated with Glutathione Sepharose 4B and cell lysates (Input) and precipitates (GST pull-down) were immunoblotted (IB) with the indicated antibodies. ( F-H ) BiFC visualization of the CAV1-PKD2 interaction at the TGN. N, nucleus. Scale bars, 10 μm. (H) Quantification of BiFC signal at the TGN. SuperPlot showing the indicated cell measurements (small symbols; 22–37 cells per condition for each biological replicate) and the median value for each independent biological replicate (larger, black-outlined circles; n = 3). Each color represents a different experimental replicate. A repeated measures one-way ANOVA test was performed using Tukey’s post-hoc multiple comparison test (*, P = 0.0262; **, P = 0.0094; ****, P < 0.0001).

Article Snippet: The plasmids encoding dog CAV1 (WT and P132L)-mEGFP were generous gifts from A. Helenius (ETH Zurich, Institute of Biochemistry, Switzerland; Addgene plasmid #27704 and #27708; ).

Techniques: Expressing, Control, Membrane, Comparison, Incubation