Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, b, c, Immunostaining of MDA-MB-231 and HeLa with p85α or p110α specific antibodies show the PI3Kα enzyme in small vesicle-like structures distributed along the microtubules. Cells growing on glass coverslips were fixed and processed for immunofluorescence study using either an anti-p85α or anti-p110α antibody as described in “Methods”. The co-localization of PI3Kα vesicles with tubulin was quantified by Pearson’s r. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Immunostaining, Immunofluorescence

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, b, Effect of MAP4 knockdown in endosomal localization of PI3Kα vesicles. siRNAs targeting the 3’ UTR region of MAP4 (siMAP4#3) were used to knockdown endogenous MAP4 in MDA-MB-231 cells expressing WT or the MTBD deletion mutant of MAP4. 48–72 hours post-transfection, cells were processed for immunofluorescence study using antibodies specific for endogenous p110α and clathrin or TFR. The quantification of the colocalization of p110α and CHC/TFR by Pearson’s r was shown in Figure 5c. The images shown are the representative images of multiple reproducible experiments. Scale bar, 5 μm

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Expressing, Mutagenesis, Transfection, Immunofluorescence

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, Overexpression of MTBD impairs MAP4 association with PI3Kα. MAP4 and HA-tagged p110α were co-transfected along with empty GFP vector or GFP-MTBD or GFP-C-tail into Cos-7 cells. 24–48 hrs post-transfection, p110α were immunoprecipitated and co-immunoprecipitated MAP4 examined by immunoblotting. Error bars denote mean±SD; n=4 independent experiments

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, in vivo association of PI3Kα with MAP4. Antibodies specific to p110α or p110β or p85α were used to immunoprecipitate wild type (MDA-MB-231) or mutant forms of PI3Kα (T47D and Cal51) and co-immunoprecipitated MAP4 examined by immunoblotting. The immunoblot shown is the representative of multiple experiments.

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: In Vivo, Mutagenesis, Immunoprecipitation, Western Blot

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, MAP4 loss affects PI3Kα vesicle distribution along microtubules. Three different individual siRNAs were used for MAP4 knockdown in MDA-MB-231 cells followed by immunofluorescence staining. The co-localization of p110α vesicles and tubulin were quantified by Pearson’s r. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Immunofluorescence, Staining

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, Immunostaining of mutant PI3Kα expressing Cal51 and T47D cells with p85α or p110α specific antibodies. PI3Kα were distributed in small vesicle-like structures and its co-localization with microtubules was quantified by Pearson’s r. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Mutagenesis, Expressing, Immunostaining

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, Effect of PI3Kα knockdown in EGF stimulated Akt activation. MDA-MB-231 cells were individually transfected with three different siRNAs specific for p110α. 48–72 hours post-transfection, cells were stimulated with EGF and activated Akt was examined by immunoblotting. Error bars denote mean±SD; n=3 independent experiments

b, c, d, Expression of HA-tagged p110α rescue the effect on endogenous p110α knockdown in EGF stimulated Akt activation. The siRNAs specific to 3’ UTR (sip110α III) wereused to knockdown p110α in mock or HA-p110α expressing cells. 48–72 hours post-transfection, cells were stimulated with EGF to examine the activation level of Akt by immunoblotting or immunofluorescence microscopy. Scale bar, 5 μm; Error bars denote mean±SD; n=4 independent experiments (b), n=30 cells from representative experiments (c)

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Activation Assay, Membrane, Transfection, Western Blot, Expressing, Immunofluorescence, Microscopy

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, b, PLA shows induced association of MAP4 and IQGAP1 and that they are localized with EGFR. MDA-MB-231 cells were stimulated with EGF and processed for MAP4-IQGAP1 PLA followed by immunostaining with EGFR. The image shown is the representative images of multiple reproducible experiments. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

c, d, e, MAP4 knockdown demonstrated by immunofluorescence study and immunoblotting. Three different siRNAs were used individually to knockdown MAP4 in MDA-MB-231 cells. 48–72 hours after siRNA transfection, cells were processed for immunofluorescence study using an antibody specific to MAP4 and tubulin. MAP4 knockdown was also shown by immunoblotting. The image and blot shown is the representative of reproducible experiments. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

f, g, Localization of PI3Kα vesicles along microtubules in WT and MTBD deletion mutant of MAP4 expressing cells after knocking down endogenous MAP4. As described above, siRNAs targeting 3’ UTR region of MAP4 were used to knockdown endogenous MAP4. Cells were processed for immunofluorescence study using antibodies specific for p110α and tubulin. Distribution of PI3Kα vesicles along microtubules was quantified. n=30 cells from representative experiments

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Expressing, Mutagenesis, Immunostaining, Immunofluorescence, Western Blot, Transfection

Journal: Nature cell biology

Article Title: Phosphatidylinositol 3-kinase Signaling is Spatially Organized at Endosomal Compartments by Microtubule-associated Protein 4

doi: 10.1038/s41556-020-00596-4

Figure Lengend Snippet: a, b, Coomassie staining of proteins co-immunoprecipitated with PI3Kα antibody (anti-p85α and anti-p110α antibodies used together) showed a distinct band above 170 kDa. Mass-spectrometry analysis of the isolated protein band revealed it as microtubule-associated protein 4 (MAP4). The image shown is the representative images of reproducible experiments. Arrow in image indicates the band of interest for mass spectrometry analysis.

c, PI3Kα in small vesicles distribute along MAP4 that mimics microtubules in different cell types. Immunofluorescence study was performed in different cell types using mouse anti-MAP4 and rabbit anti-p110α or p85α antibodies. The image shown is the representative images of reproducible experiments. Scale bar, 5 μm

d, Amino acid alignment of MAP4 MTBD along with that of Tau and MAP2. All four MTBD repeats of MAP4 (MTBD I- MTBD IV) and that of Tau and MAP2 (other microtubule-associated proteins expressed in neuronal cells) show highly similar amino acid order and microtubule-binding motif.

e, Representative MST binding affinity graphs for interaction between His-PI3Kα and GST-MAP4 proteins. Unprocessed_Gel_Image_Extended_Data_Fig5

Article Snippet: The beads were washed three times with binding buffer before eluting any bound His-PI3Kα or His-tagged domains of p110α by a 2× SDS sample buffer followed by immunoblotting using anti-His or anti-PI3Kα antibodies (#MAB050H, R&D systems; #ab40776, Abcam; #4292, Cell Signaling).

Techniques: Staining, Immunoprecipitation, Mass Spectrometry, Isolation, Immunofluorescence, Binding Assay