Fig. 4. Suppression of Beclin expression impairs macroautophagy as measured by sequestration of LDH. Control or Beclin KD cells were incubated in DMEM + 10% FBS (normal) or HBSS (starved) for 4 hours. Cells were lysed and fractionated as described in the Materials and Methods. LDH was assayed by immunoblotting aliquots of the cell lysate, and the pellet fraction was recovered after centrifugation through a metrizamide/sucrose cushion. LDH sequestered in the pellet fraction was expressed as a ratio to the total LDH in the cell lysate. The results are derived from separate fractionations of cells from three parallel cultures (mean ± s.e.).