FLAG-LHR HEK293 stable cells were transfected with an siRNA library for 96 h and stimulated with LH for 5 min (3 nM LH for Screen 1 (A) and 10 nM LH for Screen 2 (B)). Intracellular cAMP levels were measured from cell lysates by a TR-FRET cAMP assay. Controls included a non-targeting control (NTC) siRNA, and siRNA for GNAS (the essential mediator of Gas-coupled signaling), the VEE proteins APPL1 and GIPC1, and beta-arrestin-2 (ARRB2). Responses were normalized to the NTC value per each replicate, transformed to log2 and the plot is ranked from high cAMP (gray) to low cAMP (yellow). Hits found in both Screens 1 and 2 are shown as gray and yellow circles; hits found in either Screen 1 or 2 are shown as gray/black and yellow/black circles. Hits which exhibited negative effects on cell morphology and/or viability after knock-down are shown as discarded points (cross). The most changed hits present in both screens were RAP2B and RAB38. Data represent mean ± SEM (n= 3).
