JCS021006 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Quantification of the relative expression and ECM retention of mRFPo and mRFPovTSP-1C in the insoluble ECM of A549 cells. Each column represents the mean from three independent experiments. Bars indicate s.e.m. Inset panel demonstrates the retention of mRFPovTSP-1C in a representative array of ECM puncta. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. (A) Gel filtration chromatograms of 6His-Myc-type3_5-7GCT and its VGD and AAA mutants. 0.5 mg aliquots of the proteins purified by affinity chromatography from the conditioned medium of episomally transfected 293-EBNA cells were injected onto a 24 ml S200 column run in 20 mM Na-HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl₂ at a flow rate of 0.5 ml/minute. The peak at ∼8 ml corresponds to the void volume of the column and contains aggregated protein; the peak at ∼14 ml corresponds to monomeric type3_5-7GCT protein (molecular mass 44 kDa + one N-linked glycan; bovine serum albumin, a 66 kDa globular protein, elutes at 14.3 ml). (B) Near-UV circular dichroism (CD) spectra of His-Myc-tagged type3_5-7GCT and its VGD and AAA mutants. CD spectra were recorded from proteins at ∼5 µM concentration at 25°C in 0.1 mm quartz cuvettes on a Applied Photophysics Chirascan instrument.

• Supplemental Figure S3 -

  Fig. S3. Wild-type mRFPovTSP-1C or its VGD or AAA point mutants were expressed in COS-7 cells for 48 hours and collected from conditioned media on Talon metal affinity resin. Samples were analysed by SDS-PAGE and immunoblotting, under reducing (R) or non-reducing (NR) conditions. Molecular mass markers are in kDa.