Mcklh

A synthetic peptide (CSRSR(pThr)P(pSer)LP(pThr)PPTREPKK) corresponding to amino acids 208–225 in the 2N/4R human tau isoform with residues Thr212, Ser214 and Thr217 phosphorylated was used for immunization. This peptide was chosen as it contains all 3 phosphorylated amino acid residues with additional adjacent residues allowing for appropriate size for the immunization peptide. The peptide included an added Cys residue at the amino-terminus that allowed for conjugation to inject maleimide-activated mariculture keyhole limpet hemocyanin (mcKLH) (Thermo Scientific, Waltham, MA, USA). All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. The peptide–KLH conjugate was used to immunize female BALB/c mice (Jackson Laboratory, Bar Harbor, ME, USA) as previously described [75 (link)]. Spleens from the mice were harvested, and the white blood cells were fused with mouse myeloma cells (Sp2/O-Ag14; ATCC, Manassas, VA, USA). Hybridoma clones were selected using HAT supplement (Sigma Aldrich, St. Louis, MO, USA) and the surviving clones were initially screened for reactivity by enzyme-linked immunosorbent assay (ELISA) using the immunization peptide, but further assessed using a series of peptides and recombinant tau proteins, as described below.

All procedures related to animals were approved by Tsinghua Institutional Animal Care and Use Committee (Animal Protocol No.17-CC2). SGK1 pS422 and pT256 antibodies were made to synthetic phosphopeptides corresponding to mouse SGK1 amino acid residues 416-429 (EAFLGFpSYAPPVDSC) and residues 249-262 (EHNGTTSpTFCGTPEC) with an additional cysteine in the N terminus. These peptides were conjugated to keyhole limpet hemocyanin (mcKLH) (#77666, Thermo Fisher Scientific) and used as antigens for immunization of New Zealand White rabbits. Antibodies recognizing nonphospho-SGK1 were subtracted from immunized sera by passing through corresponding unphosphorylated peptide (same sequence as above without phosphorylation) chromatographic columns. Phospho-antibodies were further affinity purified from the subtracted fraction on phosphopeptide chromatographic columns (#20401; Thermo Fisher Scientific). ELISA and western blots were performed to assess the antibody specificity. Lamda PP (#P0753S, NEB) was used to dephosphorylate SGK1 proteins according to manufacturer’s instructions. FAM122A phospho-S37 antibody was generated by immunizing rabbits with synthetic phosphopeptides corresponding to human FAM122A amino acid residues 34-40 (RSNpSAPL) (Dia-An Biotechnology).