In vivo kinase assay for determining the kinase targets of IRE1
 We grew 10X150 cm2 dishes for each cell type used.
o HEK293frt parental cells
o HEK293frt with IRE1(I642G)-flag
o HEK293T-REX cells with dox inducible IRE1-tm-3F6H overexpression
 We used 10 nM Dox for 24 hrs to induce IRE1 overexpression to comparable levels to HEK293frt cells
overexpressing IRE1(I642G)-flag
 We used Tunicamycin at 2.5 µg/ml for 4 hrs or Palmitate at 1mM for 12 hrs as an ER-stressor.
 Initially, we washed the cell pellet with 2-5 ml of wash buffer without ATP and analogues.
 We spin the cells down for 15 min at 1500 rpm at 4 oC and carefully discarded the supernatant.
 We then add the permeabilization buffer with 30 µg/ml digitonin (Sigma, D141) to the cells at 3X cell
volume and resuspend the cells in this buffer, then rotate the cells for 10 min at 4oC to make sure that
digitonin is equally distributed.
 We spin the cells down at 1500 rpm for 10 min at 4oC.
 We discard the supernatant and add kinase buffer at 2.5X cell volume.
 We performed the kinase reaction at 30 oC for 1 hr shaking rigorously ( 950 rpm at a thermo-shaker).
 We stopped the kinase reaction adding the 2X lysis buffer with 20 mM EDTA and 1% NP-40 end
concentration at 2.5X cell volume.
 We then lysed the cells by 5 sec of vortexing for 10 min with 5 sec of intervals.
 We centrifuged the cell debris for 15 min at highest speed at table top centrifuge at 4oC.
 We took the supernatant and flash froze it at liquid Nitrogen.
 We took 20 µl of each reaction to measure the protein concentration by Bradford.
In vivo Wash Buffer (without detergent)
Stock
Final cons.
Hepes pH 7.4
250mM
20 mM
KoAC
1000mM
50mM
MnCl2
100mM
1mM
MgCl2
500mM
1mM
Na2Mao4
25mM
1mM
NaF
250mM
2mM
H2O
for 10 ml
800ul
500ul
100ul
20ul
400ul
80ul
8100ml
Permeabilization buffer:
Wash buffer + 30 µg/ml Digitonin
In vivo Kinase Buffer (without detergent)
Stock
Final cons.
for 10 ml
ATP
25 mM
100 µM
GTP
100 mM
1 mM
N6-Fu-ATP-gS 10 mM
100 µM
Hepes pH 7.4
250mM
20 mM
800ul
KoAC
1000mM
50mM
500ul
MnCl2
100mM
1mM
100ul
MgCl2
500mM
1mM
20ul
Na2Mao4
25mM
1mM
400ul
NaF
250mM
2mM
80ul
H2O
8100ml
** Added fresh phosphatase and protease inhibitor.
2XLysis Buffer
Stock
1M
5M
20%
Final cons.
50 mM
300mM
1mM
Hepes pH 7.4
NaCl
NP-40
H2O
** Added fresh phosphatase inhibitor.
G954 Kinase assay with only Tm treated cells:
o HEK293frt parental cells, 10 dishes
o HEK293frt with IRE1(I642G)-flag, 10 dishes
o HEK293T-REX cells with dox inducible IRE1-tm-3F6H overexpression, 10 dishes
o Cells are treated with 2.5 µg/ml Tunicamycin for 4 hrs.
Volume of the cell pellet:
o HEK293frt parental cells: around 750 µl
o HEK293frt with IRE1(I642G)-flag: around 500 µl
o HEK293T-REX cells with IRE1-tm-3F6H: around 400 µl
Protein concentration and total frozen sample volume:
HEK293frt parental+ DMSO: 3.9 mg/ml,
HEK293frt parental+ TM: 4.1 mg/ml
HEK293frt with IRE1(I642G)-flag+DMSO: 2.6 mg/ml
HEK293frt with IRE1(I642G)-flag + TM: 2.8 mg/ml,
HEK293T-REX cells with IRE1-tm-3F6H+ DMSO: 3.4 mg/ml
HEK293T-REX cells with IRE1-tm-3F6H+ TM: 3.0 mg/ml
G956 Kinase assay with only Palmitate treated cells:
o HEK293frt parental cells, 10 dishes
o HEK293frt with IRE1(I642G)-flag, 10 dishes
o HEK293T-REX cells with dox inducible IRE1-tm-3F6H overexpression, 10 dishes
o Cells are treated with 1 mM Palmitate for 12 hrs.
Protein concentration and total frozen sample volume:
HEK293frt parental+ ETOH: 2.9 mg/ml,
HEK293frt parental+ PA: 3.0 mg/ml
HEK293frt with IRE1(I642G)-flag+ETOH: 3.1 mg/ml
HEK293frt with IRE1(I642G)-flag + PA: 2.8 mg/ml,
HEK293T-REX cells with IRE1-tm-3F6H+ ETOH: 4.2 mg/ml
HEK293T-REX cells with IRE1-tm-3F6H+ PA: 3.0 mg/ml
G958 Kinase assay with both TM and Palmitate treated cells:
HEK293frt parental+ DMSO: 3.9 mg/ml,
HEK293frt parental+ TM: 3.7 mg/ml
HEK293frt with IRE1(I642G)-flag+DMSO: 2.5mg/ml
HEK293frt with IRE1(I642G)-flag + TM: 2.1 mg/ml,
HEK293T-REX cells with IRE1-tm-3F6H+ DMSO: 2.2 mg/ml
HEK293T-REX cells with IRE1-tm-3F6H+ TM: 2.3 mg/ml
HEK293frt parental+ ETOH: 5.7 mg/ml,
HEK293frt parental+ PA: 4.2 mg/ml
HEK293frt with IRE1(I642G)-flag+ETOH: 3.2 mg/ml
HEK293frt with IRE1(I642G)-flag + PA: 3.5 mg/ml,
HEK293T-REX cells with IRE1-tm-3F6H+ ETOH: 5.0mg/ml
HEK293T-REX cells with IRE1-tm-3F6H+ PA: 3.4 mg/ml