8 wells chamber slides

Stably transfected HK-2 cells expressing RFP-tagged cystinosin-LKG were grown in the 8 wells Chamber Slides (BD Falcon). Cells were fixed in 4% paraformaldehyde for 10 min at 4°C, permeabilized with 0.05% Triton X-100 for 5 min at 4°C, blocked with 1% FBS in PBS for 1 h at room temperature, and stained with primary antibody diluted 1:100 for 1 h at 4°C, followed by two 10 min washes with PBS and incubation with FITC-conjugated anti-mouse IgG antibody for 30 min at 4°C. Lysosomes were stained with anti-LAMP-2 or anti-LAMP-1 mAb (Santa Cruz Biotechnology), endoplasmic reticulum (ER) with anti-PDI mAb (Abcam), Golgi with anti-GM130 mAb (BD Biosciences).
HK-2 transfected with cystinosin-LKG and ΔSSLKG conjugated with RFP or V5His were fixed in 4% paraformaldehyde for 10 min at 4°C, permeabilized 30 min at room temperature with 0.05% saponin, 0.05% BSA, 50 mM NH₄Cl, blocked with 1% BSA in PBS for 1 h, and stained with anti-RFP (Abcam) or anti-His Tag, clone HIS.H8 (Millipore) diluted 1:200 for 1 h, incubated with FITC-conjugated anti-rabbit or anti-mouse IgG antibody respectively, in alternated washing steps with PBS.

Cells were seeded for 16–24 h into sterile 8-wells Chamber Slides (Falcon). After treatments, cells were fixed with 2% paraformaldehyde for 10 min, permeabilized in iced methanol and incubated with 3% BSA for 30 min (Sigma #A2153). After incubating with Ab against cardiac myosin, troponin T, c-Myc, KLF-4 and Nanog (see Table 1) at 4°C for 18 h, cells were washed with PBS followed by incubation with Alexa 488 anti-rabbit secondary antibody (1:500, Molecular Probes) and DAPI 1:1000 for 20 min. Coverslips were mounted in ProLong® Gold Antifade reagent (Life technologies #P36930) and examined using a confocal microscope Leica PCS SP5.