3. Peptides: use the stocks, which has been described previously.
4. Instruments used: VP-ITC, ITC200, or PEAQITC.
2.4 Protein Fragment
Complementation
Assay (NanoBiT)
1. NanoBiT PPI MCS starter system (Promega). Full length
RSK1 is cloned into the N-terminal-tagged SmBiT vector
(pBiT2.1-N[TK/SmBiT]), and the PDZ domain containing
interaction partners are cloned into the N-terminal-tagged
LgBiT vector (pBiT1.1-N[TK/LgBiT]).
2. HEK293T cells.
3. Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin/
amphotericin B. To prepare 250 ml, add 25 ml fetal bovine
serum and 2.5 ml penicillin/streptomycin/amphotericin B to
222.5 ml DMEM. Store at 4
C in 50 ml aliquots.
4. Phosphate buffered saline (PBS) buffer.
5. FuGene HD reagent (Promega).
6. Opti-MEM I buffer (Thermofisher).
7. CO 2 -independent medium supplemented with 2 mM L-glutamine and 1% antibiotics. To prepare 100 ml stock solution, add
1 ml penicillin/streptomycin/amphotericin B and 1 ml
200 mM L-glutamine to 98 ml CO 2 -independent medium.
Store at 4
C in 50 ml aliquots.
8. 135 μg/ml Epidermal growth factor (EGF): to prepare a stock
solution of 135 μg/ml, add 1 mg hEGF to 7.4 ml Milli-Q
sterile water. Store at À20
C in 0.5 ml aliquots.
9. Nano-Glo reagent (Promega).
10. White TC-treated 96-well plates.
11. Plate reader.
3 Methods
3.1 In Vitro
Phosphorylation
of RSK1 683–735
1. Add 50 μl of ATP and 500 μl of RSK1 683–735 substrate to
3.95 ml phosphorylation buffer (buffer A) and vortex it gently,
then supplement with 500 μl of RSK1-T573E 411–735 kinase
and mix it carefully (see Note 1).
2. Incubate the reaction mixture in a shaker at 37
C for 3 h.
3. Proceed with the purification of the reaction mixture by
RP-HPLC.
4. Equilibrate the RP-HPLC column with Eluent A for approximately 5 min.
5. Inject the filtered reaction mixture onto the column.
6. Wash thoroughly with Eluent A until the baseline stabilizes.
Regulation of RSK1-PDZ Domain Interactions
183
4. Instruments used: VP-ITC, ITC200, or PEAQITC.
2.4 Protein Fragment
Complementation
Assay (NanoBiT)
1. NanoBiT PPI MCS starter system (Promega). Full length
RSK1 is cloned into the N-terminal-tagged SmBiT vector
(pBiT2.1-N[TK/SmBiT]), and the PDZ domain containing
interaction partners are cloned into the N-terminal-tagged
LgBiT vector (pBiT1.1-N[TK/LgBiT]).
2. HEK293T cells.
3. Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin/
amphotericin B. To prepare 250 ml, add 25 ml fetal bovine
serum and 2.5 ml penicillin/streptomycin/amphotericin B to
222.5 ml DMEM. Store at 4
C in 50 ml aliquots.
4. Phosphate buffered saline (PBS) buffer.
5. FuGene HD reagent (Promega).
6. Opti-MEM I buffer (Thermofisher).
7. CO 2 -independent medium supplemented with 2 mM L-glutamine and 1% antibiotics. To prepare 100 ml stock solution, add
1 ml penicillin/streptomycin/amphotericin B and 1 ml
200 mM L-glutamine to 98 ml CO 2 -independent medium.
Store at 4
C in 50 ml aliquots.
8. 135 μg/ml Epidermal growth factor (EGF): to prepare a stock
solution of 135 μg/ml, add 1 mg hEGF to 7.4 ml Milli-Q
sterile water. Store at À20
C in 0.5 ml aliquots.
9. Nano-Glo reagent (Promega).
10. White TC-treated 96-well plates.
11. Plate reader.
3 Methods
3.1 In Vitro
Phosphorylation
of RSK1 683–735
1. Add 50 μl of ATP and 500 μl of RSK1 683–735 substrate to
3.95 ml phosphorylation buffer (buffer A) and vortex it gently,
then supplement with 500 μl of RSK1-T573E 411–735 kinase
and mix it carefully (see Note 1).
2. Incubate the reaction mixture in a shaker at 37
C for 3 h.
3. Proceed with the purification of the reaction mixture by
RP-HPLC.
4. Equilibrate the RP-HPLC column with Eluent A for approximately 5 min.
5. Inject the filtered reaction mixture onto the column.
6. Wash thoroughly with Eluent A until the baseline stabilizes.
Regulation of RSK1-PDZ Domain Interactions
183