Measuring Interactions Between ECM and TGFB-Like Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 224

from the ECM
of FRC osteoblasts by avian osteoclast cells. 35 S-labeled ECM was prepared from fetal
rat calvarial osteoblasts grown in six-well culture plates. Avian osteoclast precursor
cells were then seeded onto the matrix at 3

Fig. 3. Immunoprecipitation showing release of LTBP1/latent TGF β

× 10 6 cells per well. The upper panels show

the labeled LTBP1/latent TGF immunoprecipitated from the culture medium of avian

osteoclasts during days 3-4 of culture (released). The lower panels show the labeled
LTBP1/latent TGF

β

immunoprecipitated from the matrix following plasmin digestion
at the end of the experiment on day 4 (bound). Nonimmune serum controls on the right
indicate negligible precipitation of non-specific bands. Lane 1 shows a control in which
no avian osteoclasts were seeded onto the matrix. A small amount of LTBP1 is released
passively from the matrix, however, the majority remains bound. Lanes 2-5 show
matrix seeded with avian osteoclasts that were not stimulated with 1,25-dihydro-
xyvitamin D 3 . A small amount of LTBP1 was released into the medium by these cells
(lane 2). This release was inhibited by aprotinin (20

β

µ g/mL, lane 3), but not by

leupeptin (2 M , lane 5). Lanes 6-9 show matrix seeded

with avian osteoclasts that were cultured in the presence of 10 -8 M 1,25 dihydroxyvitamin
D3. These vitamin D-stimulated osteoclasts released essentially all the LTBP1/latent
TGF

µ M , lane 4) or pepstatin (1 µ

β from the matrix (lane 6). Release of LTBP1/TGF β was partially inhibited by

aprotinin (20 µ g/mL, lane 7) and leupeptin (2 µ M , lane 8), but not by pepstatin (1 µ M ,

lane 9). Note that in each case, release of LTBP1/latent TGF into the culture medium

is associated with a concomitant reduction in the amount bound to the matrix.

β

and store at -20 C, protected from light). These should be added fresh to the

media on the day of use.

°

2. Phosphate-buffered saline (PBS): dissolve 8 g NaCl, 2 g KCl, 1.44 g Na 2 HPO 4 ·

2 H 2 O, 2 g KH 2 PO 4 in 800 mL distilled water. Make volume up to 1 L. The pH
should be 7.4 without further adjustment. Filter sterilize and store at 4 ° C.

3. Radiolabeling media: to 80 mL cysteine and methionine-free DMEM (Gibco)

add 10 mL -MEM (Gibco), 5 mL dialyzed FCS (dialyze 50 mL heat-inactivated

FCS 4 times against 2 L PBS using a 12-14 kDa molecular weight cut off mem-
brane, then filter sterilize and store at -20

α

C), 2 m M LG (use 100X stock as
above), 100 U/mL P/S (use 100X stock as above), 1 mL L-methionine (commer-
cially available as 100X stock, Gibco), 100

°

µ g/mL ascorbic acid (use 100X stock