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. Author manuscript; available in PMC: 2016 May 17.
Published in final edited form as: Acta Neuropathol. 2014 May;127(5):645–665. doi: 10.1007/s00401-014-1268-0

Fig 6. The ~70 kDa protein recognized by pSer129/81A antibody is NFL phosphorylated at Ser473.

Fig 6

(A) Immunoblotting with pSer129/81A and anti-NFL antibody NR4 or Coomassie R250 stained 8% polylacrylamide gels of 2% SDS or 4M urea fractions from the cortical (CX) or cerebellar (CB) white matter of human brain. (B) pSer129/81A antibody immunoprecipitates NFL from human cortical white matter or mouse spinal cord/brain stem. Immunoprecipitation was performed as described in Material and Methods. Input, unbound, and immunoprecipitated fractions were analyzed by immunoblotting with pSer129/81A and anti-NFL antibody NR4. (C) In situ alkaline phosphatase treatment showing that the pSer129/81A-reactive ~70 kDa protein is a phospho-protein in both human and mouse brain. Nitrocellulose membranes with SDS fractions from human cortical white matter or murine SDS/urea spinal cord and brain stem were incubated without (−) or with (+) alkaline phosphatase (AP) as described in Material and Methods and immunoblotted with pSer129 or anti-NFL NR4 antibodies. (D) Immunoblotting of SDS/urea fractions from nTg/wild-type (WT) and NFL−/− mice with pSer129/81A or anti-NFL NR4 antibodies showing that the ~70 kDa protein band is NFL. (E) In vitro casein kinase II phosphorylation of recombinant wild-type (WT) and Ser473Ala NFL followed by immunoblotting with either anti-NFL antibody NR4 or pSer129/81A antibody demonstrating that pSer129/81A antibody recognized NFL phosphorylated at Ser473. GFAP, glial fibrillary acid protein; NFL, low molecular-mass neurofilament subunit; NFM, mid-sized molecular-mass NF subunit; NFH, heavy molecular-mass NF subunit.