(A) CapZβ-GFP-expressing HeLa cells were immunostained with antibodies against EEA1 (top panel) or RAB5A (lower panel). Confocal images taken along the z-axis were projected (Maximum Intensity …
Original immunoblots.
Data for Figure 1A, B.
(A) CapZβ-GFP-expressing HeLa cells were treated with or without vacuolin-1 (1 μM) for 12 hr, and were then immunostained with Red-X Phalloidin; the white dashed circles indicate the …
Original immunoblots.
Data for 1C, 1E.
(A) The expression of CapZβ in control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells. (B) Control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells were treated with EGF-488 for the indicated …
Original immunoblots.
Data for 2B–2E.
(A) Phase-contrast images of control or CapZβ-knockout HeLa cells, and the size of the cells was measured by Image J. (B) Control or CapZβ-knockout HeLa cells were plated in 24-well plates, and were …
Original immunoblots.
Data for 1B, 1D-1F, 1 H, 1I.
(A, B) HeLa cells were stably transfected with EGFP/mCherry or CapZα1-EGFP/CapZβ-mCherry constructs. These cells were either immunostained with phalloidin (A), or with an anti-RAB5A antibody (B). …
Data for 3A–3D, 3F.
(A) F-actin levels in control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells were visualized by phalloidin staining. (B) The expression of CapZα in control and CapZα1/2-knockout HeLa cells was …
Original immunoblots.
Data for 1A, 1C–1F, 1I.
(A) Control or CapZβ-knockout HeLa cells were treated with or without CK-636 (100 μM) overnight and were then immunostained with an anti-RAB5A antibody. The number and size of the early endosomes in …
Original immunoblots.
Data for 4A–4D.
(A) The control or CapZβ-knockout HeLa cells were transiently transfected with RFP-RAB5A and ARPC1B-GFP, and were then treated with or without CK-636 (100 μM) overnight, followed by confocal …
Data for 1A–1C.
(A) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to …
Original immunoblots.
Data for 5D–5G, 5J.
(A, B) Twinstrep-CapZβ with Flag-Rabaptin-5 (A) or Flag-VPS34 (B) were transfected into HEK293 cells. The cell lysates were immunoprecipitated with an anti-Flag antibody, and the pulldowns were …
Original immunoblots.
Data for 1D, 1E.
(A) The left panel shows the actin-binding motif in the CapZ structure generated from the protein data bank (PDB ID: 1IZN). Cyan color indicated CapZα structure, and brown color indicated CapZβ …
Original immunoblots.
Data for 6C–6E.
(A) HEK293 cells were transfected with Twinstrep-actin, and Myc-RAB5A, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis …
Original immunoblots.
Data for 1B.
The number and size of the early endosomes in these cells were quantified and weighted by the cell size. (B) Control or CapZβ-knockout HeLa cells were immunostained with Red-X Phalloidin; the white …
2D track analysis was performed using NIS-Elements AR software.
They were then collected at the indicated time points and subjected to western blot analysis against EGFR, ERK, pERK, and Hsp70. The p-ERKs in EGF-treated control or CapZβ-knockout cells were also …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | APRC1B (Rabbit polyclonal) | Sigma-Aldrich | HPA004832 | IF (1:100) |
Antibody | ARP2 (Rabbit polyclonal) | ProteinTech | 10922-1-AP | WB (1:2000) |
Antibody | Pan Actin (Mouse monoclonal) | Cytoskeleton | AAN02-s | IF (1:500) |
Antibody | CapZα (Mouse monoclonal) | DSHB | mAb 5B12.3 | WB (1:100) |
Antibody | CapZβ (Rabbit polyclonal) | ProteinTech | 25043-1-AP | WB (1:2000) |
Antibody | EGFR (Mouse monoclonal) | Santa Cruz | sc-373746 | WB (1:100) |
Antibody | EEA1 (Rabbit monoclonal) | CST | 3,288 | IF (1:100) |
Antibody | Flag-tag (Mouse monoclonal) | Sigma-Aldrich | F3165 | WB (1:1000) |
Antibody | HA-tag (Rat monoclonal) | Sigma-Aldrich | 11867423001 | WB (1:3000) |
Antibody | His-tag (Mouse monoclonal) | ProteinTech | 66005-1-Ig | WB (1:3000) |
Antibody | HSC70 (Mouse monoclonal) | Santa Cruz | sc-7298 | WB (1:10,000) |
Antibody | LAMP1 (Rabbit monoclonal) | CST | 9,091 | IF (1:200) |
Antibody | MYC-tag (Mouse monoclonal) | ProteinTech | 67447-1-Ig | WB (1:3000) |
Antibody | N-WASP (Rabbit polyclonal) | Novus Biologicals | NBP1-82512 | IF (0.25–2 µg/ml) |
Antibody | p16-Arc (Rabbit monoclonal) | Abcam | ab51243 | IF (1:100) |
Antibody | Rabaptin-5 (Rabbit polyclonal) | ProteinTech | 14350-1-AP | WB (1:1000) |
Antibody | Rabex-5 (Mouse monoclonal) | Santa Cruz | sc-166049 | WB (1:500) |
Antibody | StrepMAB-Classic HRP conjugate (Mouse monoclonal) | IBA | 2-1509-001 | WB (1:3000) |
Antibody | VPS8 (Rabbit polyclonal) | ProteinTech | 15079-1-AP | WB (1:1000) |
Antibody | RAB5A (Rabbit polyclonal) | ProteinTech | 11947-1-AP | WB (1:1000) |
Antibody | RAB5A (Rabbit monoclonal) | CST | 3547 | IF (1:200) |
Antibody | RAB5A (Mouse monoclonal) | CST | 46449 | IF (1:200)WB (1:1000) |
Chemical compound, drug | CK-636 | Selleck | S7497 | |
Chemical compound, drug | Latrunculin A | Santa Cruz | sc-202691 | |
Chemical compound, drug | EGF | Bio-Rad | PHP030A | |
Chemical compound, drug | GDP-β-S | Sigma-Aldrich | G7637 | |
Chemical compound, drug | GTP-γ-S | Jena Bioscience | NU-412-2 | |
Other | Transferrin-Alexa Fluor 594 | Invitrogen | T13343 | (25 μg/mL) |
Other | EGF-Alexa Fluor 488 | Invitrogen | E13345 | (1 μg/ml) |
Other | Phalloidin-iFluor 647 | Abcam | ab176759 | (1:1000) in 30 μl stock solution |
Other | Texas Red-X Phalloidin | Invitrogen | T7471 | (1:40) in 1.5 ml stock solution |
Cell line (Homo sapiens) | HeLa cells | ATCC | CRL-CCL-2 | |
Cell line (H. sapiens) | HEK293 cells | ATCC | CRL-1573 |
pCDNA3.1-Flag-Rabaptin-5 | pRK5-mRFP-FYVE | pCDNA3.1-HA-Rabex-5 |
pCDNA3.1-MYC-RAB5A | plenti-CapZβ-GFP | pCDNA3.1-CapZβ-His6 |
pRP-CapZβ-TwinStrep | pGEX-4-R5BD | pCDNA3.1-Flag-VPS34 |
pCDNA3.1-Twinstrep-RAB5A | iRFP-FRB-RAB5A | CFP-FKBP-CapZβ |
mRFP-RAB5A | pAGW-GFP-RAB5A | plenti-CapZβ-mcherry |
pGEX4T-1-RAB5A | pGEX4T-1 | plenti-Flag-Rabex-5 |
Plenti-GFP-ML1N2* | plenti-CapZβ | pRSFDuet-1-CapZβ/CapZα1 |
plenti-CapZβ-Twinstrep | psPAX2 | pMD2.G |
pGEX4T-1-Rabaptin-5 | pCDNA3.1-β-actin-Twinstrep | plenti-CapZβ(1-147)-Twinstrep |
plenti-CapZβ(148-277)-Twinstrep | mCherry-ARPC1B |