MA1-064

antibody from Invitrogen Antibodies

Targeting: AP2A1 ADTAA, CLAPA1

Provider product page for MA1-064

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [54]
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Validations
Immunocytochemistry [4]
Other assay [11]

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Product number: MA1-064 - Provider product page
Provider: Invitrogen Antibodies
Product name: alpha Adaptin Monoclonal Antibody (AP6)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA1-064 detects assembly polypeptide 2 (AP2) from a wide variety of mammalian sources, including human, hamster, monkey, bovine, rat, and mouse. It recognizes the products of both alpha-adaptin genes, alpha A and alpha C as well as an alternatively spliced isoform of alpha adaptin found in neurons. MA1-064 has been successfully used in Western blot, immunofluorescence, immunocytochemistry, and immunoprecipitation procedures. By Western blot, this antibody detects an ~100 kDa doublet representing the two isoforms of alpha adaptin in rat brain (alpha C is the dominant form seen) Microinjection of cells with MA1-064 results in variable blockade of endocytosis. The MA1-064 immunogen is purified adaptors.
Reactivity: Human, Mouse, Rat, Bovine, Hamster
Host: Mouse
Isotype: IgG
Antibody clone number: AP6
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

AP2A1 protein structure - MA1-064 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of alpha Adaptin using alpha Adaptin Monoclonal Antibody (AP6) (Product # MA1-064) shows staining in Hela Cells. alpha Adaptin (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing alpha Adaptin (Product # MA1-064) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of alpha Adaptin using alpha Adaptin Monoclonal Antibody (AP6) (Product # MA1-064) shows staining in MCF-7 Cells. alpha Adaptin (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing alpha Adaptin (Product # MA1-064) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of alpha Adaptin using alpha Adaptin Monoclonal Antibody (AP6) (Product # MA1-064) shows staining in U251 Cells. alpha Adaptin (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing alpha Adaptin (Product # MA1-064) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of alpha Adaptin was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with alpha Adaptin (AP6) Mouse Monoclonal Antibody (Product # MA1-064) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing punctuated cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Cellular effects of LRRK2 on AP2. (a) We expressed and immunoprecipitated GFP or GFP-tagged AP2beta subunit followed by immunoblot for endogenous members of the AP2 complex and for endogenous LRRK2. Markers on the left of all blots are in kilodaltons (b) R1441C LRRK2 lowers AP2mu phosphorylation. Mock transfected, or HEK293 cells overexpressing WT or R1441C LRRK2 were treated with pcalyculinA and probed for LRRK2, total and T156 phosphorylated AP2mu as indicated. Markers on the left of all blots are in kilodaltons. A significant decrease of phospho-AP2mu/total AP2 was observed in cells expressing R1441C LRRK2 ( n = 9; AdjP, 0.0127, *P < .05; One-way ANOVA with Dunnett's multiple comparisons test). (c) Representative images of HEK293 cells stained for endogenous AP2alpha following either mock, LRRK2 WT or R1441C transfection. Scale bar: 5 muM. (d) Integrated density/area and mean vesicle size of AP2alpha was determined in areas positive for LRRK2 expression (ns, not significant; *, P < .05; **, P < .01; One-way ANOVA with Dunnett's multiple comparisons test) (e) Representative images of HEK293 cells expressing either WT or R1441C LRRK2 and assayed for transferrin uptake at T10. (f) Time course quantification of internalised transferrin at 2,5 and 10 min. Expression of the R1441C mutant resulted in a significant decrease in internalised transferrin relative to WT (****, P < .0001; Unpaired two-tailed t -test with Welch's correction for unequal variance). (g) Representati

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 The extent of Clathrin/APP and AP2/APP colocalization is reduced in NSCs from patients with AD . Confocal microscopy analysis of double staining using rabbit anti-APP and mouse anti-AP2 (A) and mouse anti-Clathrin (B) in control (C18) and AD neurons with PS1 mutations (L286V; M146L; A246E). Colocalization analysis is reported in (C) . The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in at least five separate fields per slide in 10 different slides for each NSCs. The data are expressed as mean +- SEM. Scale bars are 5 mum for APP/AP2 and 6 mum for APP/Clathrin colocalization. Scale bars in high-resolution pictures are 5 mum. Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Tyr kinase inhibitors restore APP colocalization with Clathrin and AP2 in NSCs from patients with AD . (A) IP analysis of control and PS1 neurons that were exposed, or not exposed, to Sunitinib and PP2. Control C18 and AD samples (LV, ML, AE) were immunoprecipitated with anti-pTyr agarose conjugated antibody (4G10) and analyzed with rabbit anti-APP (clone Y188). The image is representative of four independent experiments. Quantification is reported in (B) . Data were normalized with pTyr pulled down levels (input) and expressed as % of C18. Statistically significant differences were calculated by one-way ANOVA for repeated measures followed by Tukey's post hoc test for multiple comparisons. Confocal microscopy analysis of double staining using rabbit anti-APP and mouse anti-AP2 (C) and mouse anti-Clathrin (D) in controls and in neurons carrying L286V or M146L mutation on PS1 gene following exposure to the Tyr kinase inhibitor Sunitinib. The panels are representative of four different experiments performed in triplicate. (E) reports quantitative analysis of APP colocalization to AP2 and Clathrin after 12 h of exposure to Sunitinib. (E) also reports colocalization analysis after PP2 exposure. The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in five separate fields per slide in five different slides for each experimental point. The data are expressed as mean +- SEM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Tyr phosphatase inhibitors reduce APP colocalization with Clathrin and AP2 and increase phosphorylation of APP Tyr in control neurons . Confocal microscopy analysis of double staining with rabbit anti-APP and mouse anti-AP2 (A) or mouse anti-Clathrin (B) in C18, L286V, and M146L neurons before and after Tyr phosphatase inhibitor (TC2153) exposure. The panels are representative of five different experiments performed in duplicate. (C) Colocalization analysis of APP to AP2 and Clathrin following incubation with TC2153 and BVT948 (BVT) inhibitors in C18 neurons and in AD neurons. The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in five separate fields per slide in four different slides for each experimental point. The data are expressed as mean +- SEM. Scale bars 6 mum and 4 mum. Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test. (D,E) . IP analysis of C18 (D) and AD neurons (E) before and after exposure to TC2153 and BVT948 (TC, BVT). Samples were immunoprecipitated with anti-pTyr agarose conjugated antibody (4G10) and analyzed with rabbit anti-APP (clone Y188). Densitometric analysis is reported in (F) . Data were normalized with IgG levels and expressed as % of C18. Statistically significant differences were calculated using Student's t -test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 APP/AP2 and APP/Clathrin binding analysis in cortical tissues and fibroblasts of Gottingen minipigs with a PS1 M146I mutation in the PS1 gene . (A) CoIP analysis of cortical tissues and fibroblasts from control (Ctrl) and PS1 M146I minipigs. Samples were immunoprecipitated with rabbit anti-APP and analyzed with mouse anti-Clathrin (Clath) and mouse anti-AP2. (B) WB analysis of total lysate from controls (WT and Ctrl) and PS1 M146I minipigs. Densitometric analysis is reported in (C) . Data were normalized to the corresponding beta-actin values and expressed as % of Ctrl. Data from CoIP samples were normalized to the corresponding APP input amount and expressed as % of Ctrl. Statistically significant differences were calculated using Student's t -test. (D,E) Confocal microscopy analysis of double staining with rabbit anti-APP and mouse anti-AP2 (D) and mouse anti-Clathrin (E) in fibroblasts from Ctrl and PS1M146I minipigs in the presence of TC2153 or Sunitinib. The panels are representative of five different experiments performed in duplicate in Ctrl and PS1 fibroblasts from two independent control (Ctrl 1-2) and three PS1 (PS1a-c) minipigs. Colocalization analysis is reported in (F) . The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in six separate fields per slide in four different slides for each cell line. Scale bars 1 muM and 5 muM in high magnification. Statis

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 APP binding to Clathrin and AP2 is compromised in AD neurons. (A) ELISA analysis for secreted Abeta42 (Ab42) and Abeta40 (Ab40) levels from controls (C18 and C16) and AD neurons carrying mutations in the PS1 gene (L286V, M146L, and A246E) after 4 and 6 weeks in culture. Abeta levels were assessed from media after 24 h of plating. Data are expressed as pg/mug (pg/ug) of total protein. Each data point is the mean +- SEM of triplicate determinations of four independent experiments. (B) Co-Immunoprecipitation (CoIP) analysis from controls (C18, C16) and AD neurons (LV, ML, AE). Samples were immunoprecipitated with rabbit anti-APP (clone Y188) and analyzed with mouse anti-Clathrin (clone X22), mouse anti-AP2 (clone AP6), and rabbit anti-AP1 (left panel). The right panel shows total levels of APP, Clathrin, AP2, and AP1 expression in the same samples. Densitometric analysis is reported below. Data from total lysate samples were normalized to the corresponding beta-actin values and expressed as % of C18. Data from IP samples were normalized to the corresponding APP input band and expressed as % of C18. The data are representative of five independent experiments. Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 alpha TAT1 downregulation decreased F-actin and altered alpha -adaptin distribution. ( a ) Confocal images of A549 cells visualized with Alexa Fluor 488 phalloidin (green) and immunostained with anti-vinculin antibody (red). ( b ) Confocal images of metaphase HeLa cells visualized with Alexa Fluor 488 phalloidin. Single optical section of the middle plane is shown. ( c ) pTyr397-PTK2 level determined by western blotting. The same protein extract was used in Figures 1a and 2c , therefore the same blotting of beta -Tu is presented in both figures. ( d ) Confocal images of metaphase HeLa cells immunostained with anti- alpha -adaptin antibody. Single optical section of the bottom or middle planes are shown. The signal intensity across the dashed lines is plotted in the right panel and the cartoon below indicates the relative position of the metaphase cell. Scale bar, 10 mu m.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 Cellular effects of LRRK2 on AP2. (a) We expressed and immunoprecipitated GFP or GFP-tagged AP2beta subunit followed by immunoblot for endogenous members of the AP2 complex and for endogenous LRRK2. Markers on the left of all blots are in kilodaltons (b) R1441C LRRK2 lowers AP2mu phosphorylation. Mock transfected, or HEK293 cells overexpressing WT or R1441C LRRK2 were treated with pcalyculinA and probed for LRRK2, total and T156 phosphorylated AP2mu as indicated. Markers on the left of all blots are in kilodaltons. A significant decrease of phospho-AP2mu/total AP2 was observed in cells expressing R1441C LRRK2 ( n = 9; AdjP, 0.0127, *P < .05; One-way ANOVA with Dunnett's multiple comparisons test). (c) Representative images of HEK293 cells stained for endogenous AP2alpha following either mock, LRRK2 WT or R1441C transfection. Scale bar: 5 muM. (d) Integrated density/area and mean vesicle size of AP2alpha was determined in areas positive for LRRK2 expression (ns, not significant; *, P < .05; **, P < .01; One-way ANOVA with Dunnett's multiple comparisons test) (e) Representative images of HEK293 cells expressing either WT or R1441C LRRK2 and assayed for transferrin uptake at T10. (f) Time course quantification of internalised transferrin at 2,5 and 10 min. Expression of the R1441C mutant resulted in a significant decrease in internalised transferrin relative to WT (****, P < .0001; Unpaired two-tailed t -test with Welch's correction for unequal variance). (g) Representati