Journal: bioRxiv
Article Title: Regulation of the Na-K-2Cl cotransporter NKCC2 by ubiquitylation
doi: 10.64898/2026.05.07.723577
Figure Lengend Snippet: A) After treatment of MDCKI-hNKCC2 cells with tetracycline, RT-PCR with primers against hNKCC2A and 18S rRNA (control) demonstrated robust NKCC2 mRNA expression. Immunoblotting of MDCKI-hNKCC2 cells using a flag-tag antibody demonstrated relatively low NKCC2 protein levels. B) Representative immunoblots using flag-tag or 20S proteasome (control) antibodies of MDCKI-hNKCC2A cells pretreated with 5 or 15 mM valproic acid (VpA) for 24 h prior to tetracycline (Tet) induction. C) Immunoblots of MDCKI-hNKCC2A cells treated with tetracycline combined with VpA for various time points. D) Representative immunoblots of lysate from tetracycline and VpA induced MDCKI-hNKCC2A cells grown on semi-permeable supports subjected to apical surface biotinylation. Total and biotinylated pools were blotted with flag-tag or 20S proteasome antibodies. E) Representative confocal laser micrograph (top, xy plane; bottom, xz plane) of tetracycline and VpA induced MDCKI-hNKCC2A cells grown on semi-permeable supports labeled with flag-tag antibody. F) NKCC2 was immunoprecipitated (IP) from MDCKI-hNKCC2 cells using a flag-tag antibody, with negative controls performed with or without addition of antibody or lysate. Immunoprecipiates were blotted for flag (NKCC2) or total ubiquitin (Ubi). G) NKCC2 exists as a highly complex glycosylated protein. Representative immunoblots using flag-tag and 20S proteasome (control) antibodies of lysates from tetracycline and VpA induced MDCKI-hNKCC2 cells treated with PNGase F (1 or 2 µl) or EndoH (2 or 5 µl) for 1 h at 37°C.
Article Snippet: Protein concentrations were measured using BCA assays (Thermo) and equal protein amounts were denatured in 1× denaturing buffer (New England Biolabs) for 15 min at 65 °C, chilled on ice and centrifuged for 10 sec, before deglycosylation using PNGase F (Peptide N-glycosidase F) or EndoH (Endoglycosidase H) (New England Biolabs) for 1 h using standard protocols.
Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Western Blot, FLAG-tag, Labeling, Immunoprecipitation, Ubiquitin Proteomics
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