Journal: Journal of Biological Chemistry

Article Title: A repeated triple lysine motif anchors complexes containing bone sialoprotein and the type XI collagen A1 chain involved in bone mineralization

doi: 10.1016/j.jbc.2021.100436

Figure Lengend Snippet: Figure 1. Biomineralization foci are selectively enriched in bone sialoprotein and “6b” isoform of Col11a1 chain. A, alizarin red S stained bio- mineralization foci (arrow) before laser capture. Microscopic view of BMF before capture. Bar, 50 microns. B, view of same microscopic field shown in A after laser capture of two BMF. Arrow identifies position where uppermost BMF was removed. Bar, 50 microns. C, view of “cap” after laser capture of two BMF shown in A. Arrow identifies position of uppermost BMF captured. D, western blotting on pooled laser captured BMF sample compared with total mineralized cell layer (+CL), total unmineralized cell layer (−CL), and buffer alone (buffer). Lines between lanes represent splice junctures between different gel lanes electrophoresed on the same gel. Molecular weight estimates refer to blue prestained globular standards co-electrophoresed on the same gel. BMF, biomineralization foci.

Article Snippet: Coomassie blue prestained globular protein molecular weight standards (BioRad, Inc) were coelectrophoresed with unknowns for estimation of protein mass.

Techniques: Staining, Western Blot, Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: A repeated triple lysine motif anchors complexes containing bone sialoprotein and the type XI collagen A1 chain involved in bone mineralization

doi: 10.1016/j.jbc.2021.100436

Figure Lengend Snippet: Figure 3. Co-purification of 60 kDa Col11a1 NTD “6b”-expressing fragment with full-length bone sialoprotein by anion-exchange chromatog- raphy. A, tracing of anion-exchange chromatography of EDTA extract of mineralizing UMR106-01 osteoblastic cultures. Flow-thru fractions are not shown before the start of the linear 0 to 0.6 M sodium chloride salt gradient. UV readings at 214 and 280 nm, and conductivity are plotted as a function of time and fraction number. At the end of the salt gradient, a 2 M NaCl step gradient was applied (designated by arrow). B, individual fractions were dot-blotted sequentially onto PVDF membrane and subjected to immunodetection with antibodies against Col11a1 chain NTD “6b” epitope or bone sialoprotein. C, peak fractions eluting with 2 M NaCl (#91–95) were pooled and subjected to SDS-PAGE, and the gel was either stained for MAA lectin, which identified a prominent BSP band at 90 kDa, or, the gel was western blotted with anti-BSP and anti-Col11a1 NTD “6b” epitope specific antibodies. Molecular weight estimates refer to blue prestained globular standard proteins co-electrophoresed on the same gel. BSP, bone sialoprotein; MAA, Maackia amurensis agglutinin; NTD, N-terminal domain; PVDF, polyvinylidene difluoride.

Article Snippet: Coomassie blue prestained globular protein molecular weight standards (BioRad, Inc) were coelectrophoresed with unknowns for estimation of protein mass.

Techniques: Expressing, Chromatography, Membrane, Immunodetection, SDS Page, Staining, Western Blot, Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: A repeated triple lysine motif anchors complexes containing bone sialoprotein and the type XI collagen A1 chain involved in bone mineralization

doi: 10.1016/j.jbc.2021.100436

Figure Lengend Snippet: Figure 5. Col11a1 peptide 3 binding to osteoblastic cell fractions identifies two protein ligands: bone sialoprotein and nucleolin. A, western blots identify bone sialoprotein and nucleolin. “12 pattern” SDS PAGE gel was run as noted previously (15, 31), subjected to Western blotting, and blots incubated with FAM-labeled peptide 3. Lanes represent four different cell culture conditions [mineralizing (+BGP) and nonmineralizing (−BGP, +BGP + AEBSF protease inhibitor, −BGP + AEBSF)], three different cell fractions from each condition [(cell media, EDTA extract enriched in biomineralization foci), and urea extract (cell membrane and contents)]. Line on figure indicates position of splice junction between gel lanes electrophoresed on the same gel. Molecular weight estimates are based on blue prestained globular protein standards co-electrophoresed on the same gel. B, gel lanes after electrophoresis of EDTA extract provides material for LC-MS/MS peptide mapping and Edman sequencing. EDTA extract from mineralizing UMR106-01 cultures electrophoresed on SDS- PAGE gel and stained with Coomassie brilliant blue dye. Bands at 110 kDa and 90 kDa were excised (arrows) and subjected to mass spectroscopic peptide mapping. A band at 18 kDa (arrow) was also cut out and the contents subjected to micro-Edman sequencing. Standard lane (Std): 250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa, 25 kDa, 20 kDa, and 15 kDa. See Methods for more details. C, western blot incubated with anti-nucleolin antibodies. Urea and EDTA extracts from mineralizing UMR106-01 cultures were electrophoresed on SDS-PAGE gel and blotted onto PVDF membrane. Lanes represent four different cell culture conditions [mineralizing (+BGP) and nonmineralizing (−BGP, +BGP + DEC protease inhibitor, −BGP + DEC inhibitor)]. Molecular weight estimates are based on blue stained globular protein standards co-electrophoresed on the same gel. D, purified full length calvarial bone sialoprotein binds Col11a1 NTD derived peptide 3 robustly. Bone sialoprotein was purified from calvarial bone, subjected to SDS-PAGE over a range of 1 to 10 μg protein/lane, and blotted onto PVDF membrane as described in Methods. Blots were blocked with casein and then incubated with FAM-labeled Col11a1 NTD derived peptide 3. After excess peptide was removed by washing, blots were imaged with a Fuji LS 4000 fluorescent imager. +B, plus BGP; +B + DEC, plus BGF and decanoyl-RRLL-chloromethylketone; +DEC, without BGP and with decanoyl-RRLL-chloromethylketone; and, blank, without BGP and without decanoyl-RRLL- chloromethylketone; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; BGP, β-glycerolphosphate; BSP, bone sialoprotein; FAM, 6- carboxyfluorescein; NTD, N-terminal domain; PVDF, polyvinylidene difluoride.

Article Snippet: Coomassie blue prestained globular protein molecular weight standards (BioRad, Inc) were coelectrophoresed with unknowns for estimation of protein mass.

Techniques: Binding Assay, Western Blot, SDS Page, Incubation, Labeling, Cell Culture, Protease Inhibitor, Membrane, Molecular Weight, Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Sequencing, Staining, Derivative Assay