Journal: Scientific Reports
Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis
doi: 10.1038/s41598-021-99348-z
Figure Lengend Snippet: PEA-15 regulates integrin trafficking. ( A ) Strip-resistant (internalized) biotinylated integrins were quantified by capture ELISA with antibodies against α5 integrin and expressed as a percentage of surface integrin absorbance signal. Shown is internalization after 5 min ( p = 0.2299, n = 2) and 15 min (* p < 0.05, n = 5) in the PEA-15 knock down cells compared to scramble control. Chased (recycled) biotinylated integrins were also quantified by capture ELISA. Shown is the percent α5 integrin recycled relative to pulse controls at 10 min (* p < 0.005, n = 4) and 20 min ( p = 0.5452, n = 3). Western blots show expression of α5 integrin, β1 integrin, Rab5 and Clathrin Heavy Chain, in the stable PEA-15 KD cells. ( B ) Surface biotinylated integrin α5β1 was quantified by capture ELISA (normalized absorbance to scramble control, p = 0.2439, n = 7). Degradation of surface α5 integrin was quantified over 6 h. Shown is the percent signal compared to time 0 (n = 3). ( C ) Integrin endocytosis was quantified in U87MG with two independent siRNAs (1 and 2). Shown is internalization normalized to non-target (NT) control (* p < 0.05, n = 3 for siRNA 1, n = 2 for siRNA 2). ( D ) Integrin endocytosis was measured using immunoprecipitation with antibodies against β1 integrin and detection with Western blotting with Streptavidin-HRP. Shown is a representative Western blot and internalization time course with points at 10 min ( p = 0.2425, n = 3) and 15 min (* p < 0.05, n = 3). ( E ) Surface α5β1 integrin was labeled with mouse anti-α5 antibodies in PEA-15 KD cells and scramble controls on ice. Cells were then returned to 37 °C in the presence of EGF to induce integrin trafficking, followed by fixation and immunostaining. HA-tag PEA-15 expressed in a subset of PEA-15 KD cells were co-stained with anti-HA-tag antibodies. Asterisks (*) mark transfected cells. Inset images show red-fluorescent HA-tag signal. Scale bar = 20 µm. ( F ) Integrin endocytosis was quantified in PEA-15 KD cells expressing HA-tagged PEA-15 (* p < 0.05, Scramble n = 3, PEA-15 shRNA n = 4, HA-PEA15 rescue n = 3, HA-S104A n = 3, HA-S116A n = 3).
Article Snippet: Stable knock down of PEA-15 in U87MG cells was done using lentivirally packaged shRNA pools of 3 targeting sequences against PEA-15 (sc-37485-V) or scrambled sequence control (sc-108080) using the manufacturer’s recommendations (Santa Cruz Biotechnology, Dallas, TX).
Techniques: Stripping Membranes, Enzyme-linked Immunosorbent Assay, Knockdown, Control, Western Blot, Expressing, Immunoprecipitation, Labeling, Immunostaining, Staining, Transfection, shRNA
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