Fig 1: (a) PK-15 cells expressing 3A (O99) were stained with specific anti-Sec23A or Sec31A rabbit antibodies, followed by anti-rabbit antibodies conjugated with Alexa Flour 594. Finally, the 3A (O99) protein was further identified using QA2-Dylight488. The white arrow indicates the cell expressing 3A showing downregulation of Sec31, while white arrowheads indicate those showing Sec31 dispersion. (b) PK-15 cells were transfected using an empty vector (pcDNA-3.1(+)), pcDNA-mGFP, pcDNA-3A, or pcDNA-mGFP-3A for 21 h. The cells were further incubated in either normal, 100 nM rapamycin, or starvation condition medium for 3 h. The LC3B proteins from these cell lysates were analyzed by Western blotting and quantified using the ImageJ software. (c) PK-15 cells co-expressing mCherry-LC3B with mGFP or mGFP-3A. The cells expressing mGFP in starvation condition medium for 3 h were regarded as a positive control for autophagy activation and showed much more LC3B punctae than did the non-treated cells, but there was no significant increase in mGFP-3A overexpression. Scale bar, 10 μm. **** p < 0.0001.
Fig 2: DNM2 was the protein partner of PKCα in PEVs generation. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify the relative expression level of PKCα mRNA in platelets from breast cancer patients (n = 3) and from benign patients (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Western blotting analysis of total PKCα and phosphorylated PKCα protein levels in platelets from cancer patients and benign patients. (C) Immunofluorescent staining micrographs show PKCα (green) located in the cytoplasm or membrane of platelets from a benign patient (left), while in platelets from a patient with breast cancer, most PKCα translocated from the cytoplasm to the cell membrane (right). Original magnification of the 63× oil immersion objective in confocal microscopy. (D) Western blotting analysis demonstrated the changes in total PKCα and phosphorylated PKCα protein levels in platelets treated for 30 minutes with 0.05 μM PMA, 1 μM GÖ6976, 10 μM LY333531, or 0.1% DMSO as a vehicle control. (E) Washed platelets from breast cancer patients (n=32) were treated with 0.05 μM PMA, 1 μM GÖ6976, 10 μM LY333531 or 0.1% DMSO for 2 hours. The PEVs in the four groups were then analyzed using small-particle flow cytometry based on reference beads. (*p < 0.05, **p < 0.01, ***p < 0.001). (F) Silver staining of polyacrylamide gels (upper left panel), Venn diagram (upper right panel), and mass spectrometry (MS, lower panel) assays revealed the proteins pulled down from total platelet membrane proteins by an anti-phosphorylated PKCα antibody, along with their overlapping analysis. (G) Western blotting analysis demonstrated that other proteins potentially related to DNM2, including EXOC3L4, SEC10, HEATR5B, and SEC31A, did not show significant changes in response to stimulation in the cancer samples. (H) Co-immunoprecipitation analysis demonstrated that platelet lysates immunoprecipitated with an anti-p-PKCα antibody, but not with an antibody against total PKCα, were DNM2 positive by immunoblotting. Additionally, immunoprecipitation with an anti-DNM2 antibody showed positivity for p-PKCα. (I) In vitro kinase assay. ATP was removed from the reaction system as a negative control (lane 2), and compared to the direct substrate of PKCα, malantide (a highly specific substrate for PKCα) (lane 4), used as a positive control. The first and the last lanes were molecular weight marker, indicating the approximate size of proteins in the gel. The results of lane 3 indicated that DNM2 was not a direct substrate of PKCα. (J) Western blotting analysis of DNM2 expression in platelets from cancer patients and benign patients. (K) Platelets from breast cancer patients were stained with phalloidine (red), phosphorylated PKCα (green), and DNM2 (violet). Colocalization of the three proteins was observed and photographed under a 100× oil immersion objective using a confocal microscope. White arrows indicate membrane blebbing and the budding of EVs from platelets, where the three proteins F-actin, p-PKCα, DNM2 colocalized.
Fig 3: Proteasome activity is critical for maintaining the cell-type-specific proteome(A) Replot of Figure 1G, proteins with statistically different changes in protein expression are colored by whether they are tissue-specific (red), not tissue-specific (blue), or not present in the RNA-seq database used for tissue-specificity determination (NA, not available, black).(B) Heatmap of the normalized transcript per million (nTPM) values across 50 different tissue types for the most significantly affected proteins (|log2| ≥ 2) in (A). For each protein, nTPM values were normalized to the highest nTPM value across all tissues. Color scale indicates the normalized nTPM values, with white representing lower and red representing higher values as indicated.(C–E) Immunoblots of lysate from HCT116 WT and hRpn10VWA detecting PALM3, SUSD2, CDK6, SEC31A, RAB25, S100A14, hRpn10, and β-actin.(F) Model of proteasome activity contributing to cell identity that is represented by colored spots inside squares. A cartoon of a cell with the cytosol (yellow) and nucleus (light yellow) is displayed and an aberrant proteome (represented by little color variance) for cells with hRpn10VWA proteasomes contrasted with a healthy proteome for cells with WT proteasomes.See also Tables S1 and S2.
Supplier Page from Abcam for Anti-Sec31A antibody