- Seed 3x104 HeLa cells in DMEM (PAN Biotech, P04-41450, 2730618) supplemented with 10% FBS (PAN Biotech, P03-3302), without antibiotics, in 6 well plates in 2 ml media.
- Grow for 24h cells at 37°C and 5% CO2 to 70% confluency.
- Change medium.
- Transfect cells with 60pmol ON-TARGETplus SmartPool siRNA human (Dharmacon, L-012571-00-0005, lot 170831) in 125µl 150mM NaCl added to 7.5µl Lipofectamine RNAiMax (13778030, lot 1911816) in 125µl 150mM NaCl.
- Grow cells for 24h at 37°C and 5% CO2.
- Change medium.
- Grow cells for 72h at 37°C and 5% CO2.
- Wash cells once with PBS (PAN Biotech, P04-36500, lot 3620618).
- Detach cells with 100µl trypsin per well.
- For WB the cells were once washed with PBS (PAN Biotech) before treated with 100µl of Trypsin (PAN Biotech, P10-023100, lot 6860417) per well.
- Once cells are detached add 900µl medium.
- Count cells.
- Add 40µl 1x Laemmli buffer (Roth, K929.1, lot 227259243) to 2x105 cells. Heat up to 99°C for 10min.
- Separate 10µl of the lysates on a freshly cast denaturing 12% SDS-PAGE gel in an electrophoresis chamber for 1h at 120V.
- Transfer proteins onto PVDF membrane (Merck, IPVH0010, lot K5KN7128I) with a wet blotting system for 1h at 80V.
- Block the membrane with 1x Roti Block (Roth, A151.2, lot 148270222) for 30min at RT.
- Incubation with primary
- mouse anti-CLNS1A antibody (antibodies-online, ABIN933127, lot 9832) diluted 1:500 in 1x Roti Block for 1h at RT,
- mouse anti-CLNS1A antibody (Santa Cruz, sc-271454, G3010) diluted 1:3000 in 1x Roti Block for 1h at RT, or
- rat anti tubulin antibody (YOL 1/34 gifted) diluted 1:1000 in 1x Roti Block for 1h at RT.
- Wash membrane 2x for 10min with 1x TBST.
- Incubation with secondary
- goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, 115-005-003, lot 128525) diluted 1:10000 in PBS for 45min at RT or
- goat anti-rat IgG (H+L) (Jackson ImmunoResearch, 112-035-003) diluted 1:10000 in PBS for 45min at RT.
- Wash membrane 5x for 5min with 1x TBS-T.
- Reveal protein bands using ECL (GE Healthcare, RPN3244V2, 9633722) on an LAS mini system (GE healthcare).