Abstract
Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120.
Highlights
Actin filament-associated protein-110kD (AFAP-110), originally found as a substrate and binding partner of Src kinase and protein kinase C alpha (PKCα), is an actin filament-crosslinked protein [1,2,3,4]
AFAPs expression levels were dramatically decreased in the adult brain, with a high concentration only detected in the olfactory bulb [12]
Specificity, and positive prediction values, we simultaneously used the ABCpred online serve and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of the human AFAP-120 protein [21,22], and found a 16aa-peptide sequence (SNHYKYPASAQSVTNT) was the consensus epitope predicted by both tools
Summary
Actin filament-associated protein-110kD (AFAP-110), originally found as a substrate and binding partner of Src kinase and protein kinase C alpha (PKCα), is an actin filament-crosslinked protein [1,2,3,4]. AFAP-120 contains an additional neuronal insert (NINS) near the carboxy terminus which does not disrupt the reading frame of the downstream coding sequence [11]. The staining of mouse brain sections with an antiserum against AFAPs demonstrated that AFAPs are widely expressed in embryonic and early postnatal brain regions including the cortex, forebrain, cerebellum, and olfactory bulb [12]. AFAPs expression levels were dramatically decreased in the adult brain, with a high concentration only detected in the olfactory bulb [12]. With Northern and Western forebrain, cerebellum, and olfactory bulb [12]. TThhiiss ssyynntthheettiicc ppeeppttiiddee wwaass ccoonnjjuuggaatteedd wwiitthh tthhee ccaarrrriieerr pprrootteeiinn KKLLHH aanndd wwaass tthheenn uusseedd ttoo iimmmmuunniizzee aa rraabbbbiitt ttoo pprroodduuccee tthhee aannttii--AAFFAAPP--112200 ppoollyycclloonnaall aannttiibbooddyy. RReeccooggnniizzaattiioonn ooff tthhee AAnnttii--AAFFAAPP--112200 AAnnttiibbooddyy ttoo HHuummaann NNIINNSS PPeeppttiiddee. TToo ddeeteteccttwwhehtehtehrehruhmuamnaNnINNSINcoSulcdobueldimbme uimnombluonttoebdlobtytethdebayntit-hAeFAanPt-i1-A20FaAnPti-b1o2d0y,apnltaibsmodidys, pplCaMsmVi-dFslapgC-hMuVm-aFnlaNg-IhNuSmaannd NemINpStyavnedcteomr ppCtyMvVe-cFtloargpwCeMreVt-rFanlasgfewcteerdeintrtaonHsfEecKt2e9d3iTntcoellHs EfoKr24983Th. cTehllescfeollrs4w8ehr.eTlyhseedce, lalnsdwtehreeelxytsreadct,eadnpdrothteeinexstwraecrteeidmpmroutneoinbslowtteedrewimithmaunntiobboldoitetesdagwaiitnhstaAnFtiAboPd-1ie2s0 aagnadinFsltagAtFaAgP, r-e1s2p0eacntidveFllya.gMtaega,nrweshpileec,ttivheelyra. bMbeitapnrwe-hiimlem, tuhneerasberbuitmprwe-aismumseudnaessearnuemgawtiavseucsoendtraosl. aThneegreastiuvletscionndtircoalt.eTdhteharetsouvltesr-ienxdpicreasteseddthFalatgo-vtaegrg-eexdprheussmedanFNlaIgN-tSagcgoeudldhubemiamnmNuInNoSblcootuteldd bbey iamntmi-uAnFoAbPlo-1tt2e0dabnydaannttii--AFFlaAgPa-n1t2i0boadnidesan(Ftii-gFulareg3a)n, tbiubot dnioetsb(yFipgruer-eim3)m, buuntensoetrubymp(rFei-gimurme 3u)n. e serum (Figure 3)
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