Journal: The Journal of Biological Chemistry
Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors
Figure Lengend Snippet: Expression and localization of BBS proteins are not altered in Cep290 fl/fl ;Cre + retinas. A, immunoblot analysis of Cep290 +/ fl ;Cre ? and Cep290 fl/fl ;Cre + retinal protein extracts. Each lane represents individual animals, and three animals were analyzed per genotype. Thirty μg of proteins were loaded per lane. Locations of protein standards are shown on the right. Numbers in ratio column are relative band intensities of the indicated proteins in Cep290 fl/fl ;Cre + retinas compared with those in Cep290 +/ fl ;Cre ? retinas. B, BBSome assembly is not altered in Cep290 fl/fl ;Cre + retinas. Retinal protein extracts from Cep290 +/ fl ;Cre ? and Cep290 fl/fl ;Cre + retinas were subjected to immunoprecipitation ( IP ) with anti-BBS7 antibody. Normal rabbit IgG was used as a negative control. Co-precipitated BBS proteins were detected by immunoblotting. C, immunoblot analysis of outer segment fractions isolated from Cep290 +/ fl ;Cre ? and Cep290 fl/fl ;Cre + retinas. Each lane represents individual outer segment preparations, and the results from two independent preparations are shown. Five μg of proteins were loaded per lane. Others are same as in A. D, localization of BBS8 ( green ) in Cep290 +/ fl ;Cre + and Cep290 fl/fl ;Cre + photoreceptors. Connecting cilia were labeled with anti-CETN antibody ( red ). Scale bar represents 1 μm. E, localization of LZTFL1 ( green ) in control ( Lztfl1 + lgt ), Lztfl1 gt/gt , and Cep290 fl/fl ;Cre + photoreceptors. White arrowheads indicate binding of anti-LZTFL1 antibody to cross-reacting protein(s) around the basal body. Others are same as in D .
Article Snippet: AntibodiesThe following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).
Techniques: Expressing, Immunoprecipitation, Negative Control, Isolation, Labeling, Binding Assay