Performed under reducing conditions; Samples loaded at 30 µg/lane. Membrane blocking and secondary antibody incubation performed in 5% milk/PBST. Primary antibody incubation performed in 5% BSA/PBST. Lane 1: Marker Lane 2: A431 lysate starved Lane 3: A431 lysate starved treated with 100 ng/mL of EGF for 30 min Lane 4: A431 lysate starved and pretreated for 2 hours with 10µM U0126 (ab120241) Lane 5: A431 lysate starved and pretreated for 2 hours with 30µM Ly294002 (ab120243) Primary: AKT/MAPK signaling pathway antibody cocktail at 1/250 dilution Secondary: HRP conjugated Goat anti-Rabbit secondary antibody at 1:10,000 dilution. Observed bands: RSK1p90 (pS380) band size: 83 kDa AKT1 (pS473) band size: 56 kDa ERK1 (pY204) and ERK2 (pY187) band size: 43 kDa RPS6 (pS235/236) band size: 31 kDa Rab11a band size: 24 kDa Unknown band: 130kDa

Performed under reducing conditions; Samples loaded at 30 µg/lane. Membrane blocking and secondary antibody incubation performed in 5% milk/PBST. Primary antibody incubation performed in 5% BSA/PBST. Lane 1: Marker Lane 2: A431 lysate starved Lane 3: A431 lysate starved treated with 100 ng/mL of EGF for 30 min Lane 4: A431 lysate starved and pretreated for 2 hours with 10µM U0126 (ab120241) Lane 5: A431 lysate starved and pretreated for 2 hours with 30µM Ly294002 (ab120243) Primary: AKT/MAPK signaling pathway antibody cocktail at 1/250 dilution Secondary: HRP conjugated Goat anti-Rabbit secondary antibody at 1:10,000 dilution. Observed bands: RSK1p90 (pS380) band size: 83 kDa AKT1 (pS473) band size: 56 kDa ERK1 (pY204) and ERK2 (pY187) band size: 43 kDa RPS6 (pS235/236) band size: 31 kDa Rab11a band size: 24 kDa Unknown band: 130kDa


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