Table 1.
Antibody and immunoblot protocol details
| Antibody Target | ~kDa | Cortex, μg/lane | Medulla, μg/lane | Primary Ab Supplier | Ab Host | Dilution | Time | Secondary Ab Supplier | Host and Target | Dilution | Time | Ref. |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Muscle immunoblot details | ||||||||||||
| KCC3 | 150 100 |
40, 20 | NA | A. Mercado (Mexico) | Sh | 2 μg/mL | O/N | Invitrogen | a-sheep* | 1:5,000 | 1 h | (38, 40) |
| KCC3p-T1048 | 150 | 20, 10 | NA | A. Mercado (Mexico) | Sh | 2 μg/mL | O/N | Invitrogen | a-sheep* | 1:5,000 | 1 h | (38, 40) |
| NKAα1 | 100 | 1, 0.5 | NA | Kashgarian (Yale) | Mu | 1:2,000 | O/N | Invitrogen | GAM680 | 1:5,000 | 1 h | (20) |
| NKAα2 | 100 | 5, 2.5 | NA | McDonough | Rb | 1:2,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (54) |
| NKAβ1 | 37–50 | 5, 2.5 | NA | P. Martin-Vasallo | Rb | 1:2,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (49) |
| NKAβ2 | 37–50 | 5,2.5 | NA | P. Martin-Vasallo | Rb | 1:2,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (49) |
| NKCC1 | 150 | 20, 10 | NA | DSHB (Iowa) T4 | Mu | 1:6,000 | O/N | Invitrogen | GAM680 | 1:5,000 | 1 h | (30) |
| NKCC1p-T212pT217 | 150 | 20, 10 | NA | Forbush (Yale) | Rb | 1:2,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (13) |
| OSR1 | 60–70 | 10 | NA | DSTT, Dundee | Sh | 6 μg/25 mL | 2 h | Invitrogen | DAS680 | 1:5,000 | 1 h | (55) |
| SPAK | 60–70 | 20, 10 | NA | Delpire (Vanderbilt) | Rb | 1:3,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (15, 51) |
| SPAKp-S373 OSR1p-S325 |
60–70 | 40 | NA | DSTT, Dundee | Sh | 6 μg/25 mL | 2 h | Invitrogen | DAS680 | 1:5,000 | 1 h | (15, 55) |
| Kidney immunoblot details | ||||||||||||
| AQP1 | 35 24 |
10, 5 | NA | Alpha Diagnostic Intl Inc. | Rb | 1:5,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (3) |
| AQP2 | 37 23 |
20, 10 | NA | Santa Cruz (sc. 9882) | Go | 1:500 | O/N | Invitrogen | DAG680 | 1:5,000 | 1 h | (43) |
| γENaC | 80 60 |
40, 20 | 15, 7.5 | Loffing (Zurich) | Rb | 1:10,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | (69) |
| Kir 4.1 | 40 | 40, 20 | NA | Alomone APC-035 |
Rb | 1:2,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (42) |
| NaPi2 | 85 | 40, 20 | NA | McDonough | Rb | 1:1000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | (27) |
| NCC | 150 | 40, 20 | NA | McDonough | Rb | 1:5,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | (44) |
| NCCpT53 | 150 | 40, 20 | NA | Loffing (Zurich) | Rb | 1:5,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | (60) |
| NCCpS71 | 150 | 40, 20 | NA | Loffing (Zurich) | Rb | 1:5,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | (60) |
| NHE3 | 83 | 40, 20 | 10, 5 | McDonough | Rb | 1:2,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | (79) |
| NHE3-pS552 | 83 | 5, 2.5 | 10, 5 | Santa Cruz (sc. 53962) | Mu | 1:1,000 | 2 h | Invitrogen | GAM680 | 1:5,000 | O/N | (22) |
| NKAα1 | 100 | 1, 0.5 | 1, 0.5 | Kashgarian (Yale) | Mu | 1:2,000 | O/N | Invitrogen | GAM680 | 1:5,000 | 1 h | (20) |
| NKCC | 150 | 20, 10 | 10, 5 | DSHB T4-c (Iowa) | Mu | 1:6,000 | 2 h | Invitrogen | GAM680 | 1:5,000 | O/N | (30) |
| ROMK | 50 37 |
40, 20 | NA | O. Staub (Lausanne) | Gp | 1:1,000 | 2 h | Invitrogen | GAGP 680 |
1:5,000 | 1 h | (2) |
| SPAK | 60–70 | 20, 10 | NA | Delpire (Vanderbilt) | Rb | 1:3,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h | (51) |
| SPAKp-S373 OSR1p-S325 |
60–70 | 40, 20 | NA | DSTT, Dundee | Sh | 6 μg/25 mL | 2 h | Invitrogen | DAS680 | 1:5,000 | 1 h | (55) |
| Villin | 100 | 5, 2.5 | NA | Santa Cruz (sc. 58897) | Mu | 1:2,000 | 2 h | Invitrogen | GAM680 | 1:5,000 | 1 h | (68) |
KCC3, potassium chloride cotransporter isoform 3; NKA, Na+,K+-ATPase; NKCC, Na+,K+,2Cl− cotransporter1; SPAK, Ste20/SPS-1-related proline-alanine-rich kinase; OSR1, oxidative stress-responsive kinase 1; AQP, aquaporin 1; ROMK, renal outer medullary K+ channel; ENaC, epithelial Na+ channel; NaPi2, Na+-Pi cotransporter isoform 2; NCC, Na+-Cl− cotransporter; NHE, Na+/H+ exchanger; ~kDa, apparent molecular mass determined by Bio-Rad Precision Plus Protein Dual Color Standards; Ab, antibody; Mu, mouse; Rb, rabbit; O/N, overnight; a-sheep*, anti-sheep imaged using chemiluminescence (clarity maximum: 5 min); DAS, donkey anti-sheep, DAG, donkey anti-goat; GAM, goat anti-mouse; GAR, goat anti-rabbit; GAGP, goat anti-guinea pig; NA, not assayed.