NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress- Raw data of Main Figure Panels

Figure

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Description of Files  (WCL-Whole cell lysates) IP-Immunoprecipitation

1B

1

WCL probed with Flag antibody for Flag turbo ID-αGFP6M.

2

WCL probed with GAPDH antibody.

3

WCL probed with GFP antibody for GFP-WNK1.

4

WCL full membrane probed with Streptavidin HRP antibody

1F

1

Streptavidin pulldown blot- Probed for SPAK, TSC22D4, NRBP1

2

Streptavidin pulldown blot probed for GFP-WNK1 with GFP antibody

3

Streptavidin pulldown blot probed with TSC22D2 antibody.

4

WCL probed with GFP antibody to detect GFP-WNK1.

5

WCL probed with pSPAK S371 (it picks up pOXSR1 S325).

6

WCL probed with SPAK.

7

WCL probed with TSC22D4 and NRBP1 antibody.

8

WCL probed with TSC22D2 antibody.

9

WCL probed with Tubulin antibody.

2F

1

GFP IP blot probed with Flag antibody to detect Flag-TSC22D2 and Flag-TSC22D4

2

Blots of GFP-IP (upper) & WCL (lower) probed with GFP antibody to detect GFP-TSC22D2, GFP-TSC22D4 and empty GFP 

3

WCL probed with Flag antibody to detect Flag-TSC22D2

4

WCL probed with Flag antibody to detect Flag-TSC22D4

3C

1

HA- IP blot (upper) and WCL (lower) probed with Flag antibody to detect Flag-TSC22D2

2

WCL (upper) and HA- IP blot (lower) probed with HA antibody to detect HA-NRBP1

3D

1

GFP IP blot (Upper) and WCL (Lower) probed with GFP and HA antibody to detect GFP-TSC22D2 and HA-NRBP1 

4A

1

Autoradiograph to detect NRBP1 phosphorylation by WNK 1

2

Coomassie gel for the autoradiograph in Fig 4A 1

4B

NRBP1 T232 MS spectra

4D

1

pWNK1 S382 blot

2

GST blot

3

NRBP1 blot

4

pNRBP1 Thr232 blot

4E

1

GFP IP blot probed with pNRBP1 T232 antibody

2

pNRBP1 T232 blot reprobed with GFP antibody to detect GFP-NRBP1

WCL probed with GFP antibody to detect GFP NRBP1

3

GFP IP blot probed with TSC22D4 antibody

4

WCL probed with WNK1 antibody

5

WCL probed with pSPAK S371 antibody

6

WCL probed with SPAK antibody 

7

WCL blot probed with pNCC T53/NKCC1 T212 antibody

8

WCL probed with TSC22D2 antibody

9

WCL probed with TSC22D4 and GAPDH antibody

4F

1

GFP IP blot probed with pNRBP1 T232 antibody

2

pNRBP1 blot reprobed with GFP antibody to detect GFP NRBP1

3

GFP IP blot probed with TSC22D4 antibody

4

WCL probed with WNK1, SPAK, TSC22D2, GFP and GAPDH antibody

5

WCL probed with pSPAK S371

6

WCL probed with pNCC53/NKCC1Thr212

7

WCL probed with TSC22D4 antibody (the blot in the figure is merged and was probed and developed under the same conditions)

8

WCL probed with GFP antibody to detect GFP NRBP1

5D

1

GFP IP blot probed with ELOB, pNRBP1 T232, NRBP2 and TSC22D2 antibodies

WCL probed for ELOB and TSC22D4

2

pNRBP1 T232 blot reprobed with GFP antibody to detect GFP-NRBP1

3

GFP IP blot probed with TSC22D1

4

GFP IP blot probed with SPAK

5

WCL probed with GAPDH, GFP, TSC22D4, TSC22D2, SPAK, pSPAK S371 and WNK1antibodies

GFP IP blot probed with TSC22D4 and empty GFP

6C

1

GFP IP blot probed with pNRBP1 and WNK1 antibody

2

pNRBP1 blot reprobed with GFP antibody to detect GFP-NRBP1.

GFP IP blot probed with WNK3 antibody

3

GFP IP blot probed with SPAK

4

GFP IP blot probed with TSC22D2

5

GFP IP blot probed with TSC22D4

6

GFP IP blot probed with GFP

7

WCL probed with pSPAK S373 antibody

8

WCL probed with TSC22D2, SPAK and TSC22D4

9

WCL probed with WNK1

10

WCL probed with GAPDH

7B

1

WCL probed with NRBP1(DSTT) antibody. The Bromo-tagged NRBP1 and endogenous NRBP1 is detected at 110kDa and 75kDa respectively.

2

NRBP1 blot reprobed with GAPDH and NRBP2 antibody.

3

WCL probed with pSPAK S371 antibody

4

WCL probed with SPAK antibody

7C

1

WCL probed with pSPAK S371, pNCC53/NKCC1Thr212, Bromo NRBP1, TSC22D4 and GAPDH antibodies.

2

WCL probed with WNK1 and TSC22D2 antibodies.

3

WCL probed with SPAK antibody.

7D

1

WNK1 IP blot probed with pWNK1 S382 and WNK3 antibody.

2

pWNK1 S382 blot reprobed with WNK1.

3

WCL probed with pSPAK S371, TSC22D2 and TSC22D4 antibody.

4

WCL probed with WNK1 and NRBP1 antibody.

5

WCL probed with SPAK antibody.

6

WCL probed with GAPDH antibody.

7E

1

WNK1 IP blot probed with pWNK1 S382 antibody

2

pWNK1 S382 blot reprobed with WNK1 antibody.

3

WCL probed with pSPAK S371 antibody.

4

WCL probed with SPAK antibody.

5

WCL probed with NRBP1 antibody. The band around 110kDa represents GFP NRBP1 and the band around 75kDa represents HA NRBP1.

6

WCL probed with GAPDH antibody.

8A

1

Blotting with pWNK1S382/WNK4 S335 antibody

2

pWNK4 blot reprobed with WNK4 antibody

3

Blotting with pSPAK S371/OSR1 S325 and pNRBP1 T232 antibodies

4

pSPAK S371/OXSR1 S325 blot reprobed with His antibody and pNRBP1 T232 blot reprobed with NRBP1 antibody

5

Blotting with pOXSR1 T181 antibody

8B

1

Blotting with pWNK1S382/WNK4 S335 and pSPAK S371/OXSR1 S325 antibodies

2

pWNK4 S335 blot reprobed with WNK4 antibody

3

Blotting with pOXSR1 T181 antibody

4

pOXSR1 S325 blot reprobed with His antibody

5

Blotting with NRBP1 antibody

6

Blotting with NRBP2 antibody