Anti ferritin light chain

Cell protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, blocked with 5 % nonfat milk for 1 h, and then were incubated with specific anti-ferritin light chain (1:1000, Abcam, USA), anti-β-actin (1:2000, R&D, USA), and anti-cleaved caspase 3 (1:1000, CST, USA) antibodies overnight at 4 °C. Next, the membranes were incubated with the anti-rabbit secondary antibody (1:10,000, R&D). Finally, they were detected using chemiluminescence X-ray film. The expression of β-actin was used as control.

Wild-type and PS double-knockout (PS-DK) fibroblasts were provided by Dr. Bart De Strooper (Herreman et al., 1999 (link)). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; GIBCO) containing 10% fetal bovine serum (FBS). Wild-type and PS-DK fibroblasts were lysed in RIPA buffer [10 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Non-idet P-40, 0.1% sodium dodecyl sulfate (SDS) and 0.2% sodium deoxycholate, containing a protease inhibitor cocktail (Roche)]. Equal amounts (20 μg) of protein from cell lysate were separated by SDS-PAGE in 12% gel and blotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon). The ferritin proteins were detected by Western blotting using rabbit polyclonal anti-ferritin light chain and anti-ferritin heavy chain antibodies (abcam). The blots were visualized using Super Signal Chemiluminescence solution (Wako) according to the manufacturer’s instructions. Membranes were then stripped and re-probed with anti–α-tubulin (Sigma) or anti-β-actin (Proteintec) antibodies. Quantification of protein levels was performed using Image J software (National Institutes of Health).

Total cellular proteins were extracted by solubilizing the cells in EB buffer (50 mmol/L Hepes pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 5 mmol/L EDTA, 2 mmol/L EGTA; all reagents were from Sigma-Aldrich (St. Louis, MO, USA), in the presence of 1 mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride, and a mixture of protease inhibitors. Extracts were clarified by centrifugation and normalized with the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blot detection was performed with enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA) and peroxidase conjugated secondary antibodies (Amersham, Little Chalfon, UK). The following primary antibodies were used for Western blotting (all from Cell Signaling Technology, Danvers, MA, USA, except where indicated): anti-phospho-p44/42 ERK (Thr202/Tyr204; 1:1000); anti-p44/42 ERK (1:1000); anti-phospho EGFR (Tyr1068) (1: 1000); anti-EGFR (EnzoLifeSciences, Farmingdale, NY, USA) (1:1000); anti-ferritin light chain (Abcam, Cambridge, UK) (1:1000); anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA) (1:1000).