mouse monoclonal anti human cd63 antibody  (Developmental Studies Hybridoma Bank)

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: DsRed-CD63 and CD9 localization in Rab5 endosomes in MCF7 cells. (A) MCF7 cells were transfected with each siRNA as indicated, then transfected with GFP-Rab5Q79L (green) and DsRed-CD63 (red), fixed and stained with anti-CD9 antibody (blue). The representative images of perinuclear region with enlarged endosomes are shown. Rab5 endosomes are indicated with arrowheads. Note that Rab5 endosomes 100% filled with CD63 also contained CD9 partially or 100%. In ADAP1 and ARAP1KD cells, Rab5 endosomes lost CD63 and CD9 signal. (B) The experiment in A was repeated three times. More than 10 cells were counted per experiment and more than 30 cells were classified as indicated. The percentages of each cell were shown with s.d. The culminated means of each fraction are shown on top of each bar. Control, n= 40; ADAP1KD, n= 30; ARAP1KD, n= 32. (C) The same data in B were used to make the graph of transposed x axis and classification as indicated. Scale bars: 5 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Staining, Control

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: In ADAP1 and ARAP1KD cells, CD63 incorporation in endosomes is inhibited. (A) MCF7 cells were transfected with control and ADAP1 siRNAs, and the cell lysates were subjected to western blotting. Note that ADAP1 was efficiently downregulated in ADAP1 siRNA-transfected cells compared with control siRNA-transfected cells. (B) A similar experiment for ARAP1. Note that ARAP1 was efficiently downregulated by its siRNA. (C) The siRNA-transfected cells were transfected with GFP-Rab5Q79L (green) and stained with anti-CD63 (red). The insets were enlarged and Rab5 positive endosomes are indicated with arrowheads. Note that CD63 was filled in endosomes in control cells, whereas CD63 signal was lost in ADAP1 and ARAP1KD cells. (D) The Rab5-positive endosomes were classified 100% filled, partially filled, and none with CD63. The percentage of Rab5 endosomes in each category per cell was shown. More than 15 cells were counted per experiment and the experiment was repeated four times. Error bar, standard deviation (s.d.). Control, n= 91; ADAP1KD, n =73; ARAP1KD, n= 67. Significance was calculated by two-way ANOVA with Sidak's multiple comparisons test. ns, not significant, **** P< 0.0001. Scale bars: 10 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Control, Western Blot, Staining, Standard Deviation

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: The increase in endosomes without CD63 in ADAP1 and ARAP1KD cells is not due to off-target effects. (A) MCF7 transfected control or ADAP1 siRNA were transfected with human ADAP1-HA (ADAP1 overexpression; ADAP1OE, blue) and GFP-Rab5Q79L (green). CD63 was stained with the CD63 antibody (red). The insets were enlarged and Rab5 endosomes are indicated with arrowheads. CD63 signal was seen in the control, while the signal disappeared in ADAP1KD cells. In ADAP1KD+ADAP1OE cells, the CD63 signal in the endosome was recovered. ADAP1OE cells did not show much effect on CD63 in endosomes. (B) The experiment in A was repeated three times. More than 10 cells were counted per experiment and total more than 30 cells were quantified. The percentage of endosomes without CD63 was shown with s.d. Control, n= 33; ADAP1KD, n= 34; ADAP1KD+ADAP1OE, n= 37; ADAP1OE, n= 31. The Mann–Whitney test was performed. **** P< 0.0001; ns, not significant. (C) MCF7 was transfected with siRNA ARAP1 number 2 as indicated and subjected to western blotting. (D) The cells were transfected with control or ARAP1 number 2 siRNA and immunofluorescence was performed as in A. (E) The experiment in D was repeated three times. More than 10 cells were counted per experiment and total more than 30 cells were quantified. Rab5 endosomes without CD63 was shown as in B. Control, n =35; ARAP1 number 2, n =31. The Mann–Whitney test was performed, ** P< 0.01. (F) ARAP1 number 3 siRNA was tested for western blotting as in C. (G) Immunofluorescence was performed for control and ARAP1 number 3 cells. (H) The experiment in G was repeated three times. More than 10 cells were counted per experiment and total more than 30 cells were quantified. Rab5 endosomes without CD63 were quantified. Control, n =34; ARAP1 number 3; n =37. The Mann–Whitney test was performed, **** P< 0.0001. Scale bars: 10 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Control, Over Expression, Staining, MANN-WHITNEY, Western Blot, Immunofluorescence

Journal: Biology Open

Article Title: Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies

doi: 10.1242/bio.060338

Figure Lengend Snippet: EGF and CD63 localization in Rab5 endosomes in HeLa cells. (A) HeLa cells were transfected with each siRNA as indicated, then transfected with GFP-Rab5Q79L (green), and internalized EGF-Alexa 555 for 40 min (red), fixed and stained with anti-CD63 antibody (blue). The representative images of perinuclear region with enlarged endosomes are shown. Rab5 endosomes are indicated with arrowheads. Note that Rab5-endosomes contained dotty signal of EGF, but not CD63. (B) The experiment in A was repeated three times. More than 10 cells were counted per experiment and more than 30 cells were classified as indicated. The percentages of each cell were shown with s.d. The culminated means of each fraction were shown on top of each bar. Control, n= 32; ADAP1KD, n= 32; ARAP1KD, n= 34. (C) The same data set in B were used for the graph of transposed x axis and classification as indicated. Scale bars: 5 µm.

Article Snippet: The mouse monoclonal anti-human CD63 antibody (H5C6, DSHB, IA) was kindly provided by Dr. Eiji Morita.

Techniques: Transfection, Staining, Control