261
50 mM sodium fluoride, 10 mM ß-glycerophosphate, 1× protease inhibitors (Roche, cat. no. 04693132001), 0.1% SDS
(w/v). Store at 4 °C.
4. Running buffer: 192 mM glycine, 25 mM Tris, 0.1% (w/v)
SDS in fully desalinated H 2 O.
5. PVDF membrane.
6. Transfer buffer: 125 mM glycine, 25 mM Tris, 10% (v/v)
methanol in fully desalinated H 2 O.
7. Tris-buffered saline, 0.025% (v/v) Tween-20 (TBS-T): 50 mM
Tris–HCl (pH 7.5), 150 mM NaCl in distilled H 2 O. Adjust
pH to 7.5 with HCl before adding 0.025% (v/v) Tween-20.
8. TBS-T supplemented with 5% BSA (v/v).
9. Primary antibodies (Cell Signaling Technology): AKT (pan)
(C67E7) (cat. no. 4691), Phospho-AKT (Ser473) (cat. no.
9271), Phospho-AKT (Thr308) (cat. no. 4056).
10. Secondary antibodies (Santa Cruz Biotechnology): goat antirabbit IgG-HRP (cat. no. sc-2030).
1. 1,4-diazabicyclo[2,2,2]octane (DABCO).
2. Dulbecco’s phosphate buffered saline (DPBS).
3. Immersion oil.
4. Mowiol mounting solution: 2.4 g mowiol, 6 g glycerol in
133 mM Tris–HCl, pH 8.5. Store at 4 °C.
5. Paraformaldehyde (PFA) cell fixation solution: 4% (w/v) PFA
in PBS. Store at −20 °C.
6. 12 mm round cover slips. Autoclave and store in a sealed container prior to use.
7. Fluorescence microscope with filter for green and red fluorescent proteins.
8. Microscope slides.
1. C2C12: DSMZ, Braunschweig, Germany, cat. no. ACC 565.
2. HEK293T: DSMZ, Braunschweig, Germany, cat. no. ACC 635.
3. Platinum E: Cell Biolabs, INC., San Diego, USA, cat. no.
RV-101.
4. MCF7: DSMZ, Braunschweig, Germany, cat. no. ACC 115.
HEK293T, Platinum E, and MCF7 cells are cultured in DMEM
supplemented with 10% (v/v) FCS and penicillin (100 U/ml)/
streptomycin (100 μg/ml). C2C12 cells are cultured in DMEM supplemented with 15% (v/v) FCS and 1% (v/v) penicillin/streptomycin.
Starvation was performed with DMEM supplemented with penicillin
(100 U/ml)/streptomycin (100 μg/ml) (see Note 2).
2.6 Fluorescence
Microscopy
2.7 Cell Lines
Optogenetic Control of Protein Kinases
50 mM sodium fluoride, 10 mM ß-glycerophosphate, 1× protease inhibitors (Roche, cat. no. 04693132001), 0.1% SDS
(w/v). Store at 4 °C.
4. Running buffer: 192 mM glycine, 25 mM Tris, 0.1% (w/v)
SDS in fully desalinated H 2 O.
5. PVDF membrane.
6. Transfer buffer: 125 mM glycine, 25 mM Tris, 10% (v/v)
methanol in fully desalinated H 2 O.
7. Tris-buffered saline, 0.025% (v/v) Tween-20 (TBS-T): 50 mM
Tris–HCl (pH 7.5), 150 mM NaCl in distilled H 2 O. Adjust
pH to 7.5 with HCl before adding 0.025% (v/v) Tween-20.
8. TBS-T supplemented with 5% BSA (v/v).
9. Primary antibodies (Cell Signaling Technology): AKT (pan)
(C67E7) (cat. no. 4691), Phospho-AKT (Ser473) (cat. no.
9271), Phospho-AKT (Thr308) (cat. no. 4056).
10. Secondary antibodies (Santa Cruz Biotechnology): goat antirabbit IgG-HRP (cat. no. sc-2030).
1. 1,4-diazabicyclo[2,2,2]octane (DABCO).
2. Dulbecco’s phosphate buffered saline (DPBS).
3. Immersion oil.
4. Mowiol mounting solution: 2.4 g mowiol, 6 g glycerol in
133 mM Tris–HCl, pH 8.5. Store at 4 °C.
5. Paraformaldehyde (PFA) cell fixation solution: 4% (w/v) PFA
in PBS. Store at −20 °C.
6. 12 mm round cover slips. Autoclave and store in a sealed container prior to use.
7. Fluorescence microscope with filter for green and red fluorescent proteins.
8. Microscope slides.
1. C2C12: DSMZ, Braunschweig, Germany, cat. no. ACC 565.
2. HEK293T: DSMZ, Braunschweig, Germany, cat. no. ACC 635.
3. Platinum E: Cell Biolabs, INC., San Diego, USA, cat. no.
RV-101.
4. MCF7: DSMZ, Braunschweig, Germany, cat. no. ACC 115.
HEK293T, Platinum E, and MCF7 cells are cultured in DMEM
supplemented with 10% (v/v) FCS and penicillin (100 U/ml)/
streptomycin (100 μg/ml). C2C12 cells are cultured in DMEM supplemented with 15% (v/v) FCS and 1% (v/v) penicillin/streptomycin.
Starvation was performed with DMEM supplemented with penicillin
(100 U/ml)/streptomycin (100 μg/ml) (see Note 2).
2.6 Fluorescence
Microscopy
2.7 Cell Lines
Optogenetic Control of Protein Kinases