Protein Extraction from Paraffin Embedded Sections:

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Protein Extraction from Paraffin Embedded Sections: Composition of Protein Extraction Buffer Conditions for Protein Extraction 1) 200µL RIPA buffer + 2% SDS (PI and PhospI) Ikeda, et al 100oC for 20 min (on heating block) 60oC for 2hr (water bath) Tissue lysates then centrifuged at 15,000xg for 20 min at 4oC Supernatants collected at stored @ -20 oC 2) 200µL 20mM Tris-HCl, pH 7.0 2% SDS 200mM DTT Shi, et al 3) 200µL 6M Guanidine HCl 100mM Tris HCl, pH 7.0 Jiang, et al J of Proteome Research 2007 Incubate in the lysis buffer for 72hr @ 4oC (in cold room on rocking platform) 4) 200µL Addis, et al Proteomics 2009 80oC for 2hr (with Thermomixer agitation 750rpm) 5) 1mL Qiazol (0.77g of tissue, so I rounded to 1mL of Qiazol) Protocol: Lysis of Fatty Tissue Using the TissueRuptor User-Developed Protocol: Isolation of the protein fraction from the organic phase of QIAzol Reagent-treated fatty tissue samples Protein Extraction from Paraffin Embedded Sections: Used Protocol from Qiagen for: Deparaffinization of FFPE Samples Cut Directly from a Block 5 serial sections cut at 10µm thick were used per protein extraction condition 5 conditions were used for protein extraction

First Blot: 5/24/11 Determining the best protocol for protein extraction from FFPE (formalin fixed paraffin embedded sections) Lanes 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 Short Exposure Long Exposure Lane 1: Marker Lane 2: Shed Cells (FOP sample) ~50µg Lane 3: Extraction Method 1 Lane 4: Extraction Method 2 Lane 5: Extraction Method 3 Lane 6: Extraction Method 4 Lane 7: Extraction Method 5 Loaded by volume, protein estimation was not performed

First Blot Stripped and Reprobed: 5/25/11 Determining the best protocol for protein extraction from FFPE (formalin fixed paraffin embedded sections) Lanes 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Long Exposure Short Exposure Lane 1: Marker Lane 2: Shed Cells (FOP sample) ~50µg Lane 3: Extraction Method 1 Lane 4: Extraction Method 2 Lane 5: Extraction Method 3 Lane 6: Extraction Method 4 Lane 7: Extraction Method 5 Loaded by volume, protein estimation was not performed

FFPE sample Protein Extraction Blot 1: 6/2/11 FFPE sample Protein Extraction Lanes 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Mrker Lane 1: Shed Cells (FOP sample) ~13µg Lane 2: X Lane 3: HB09-6 Lane 4: HB09-7 Lane 5: HB09-9 Lane 6: HB08-19 Lane 7: HB09-20 Lane 8: HB09-20 (6/1/2011) Mrker Lane 1: Shed Cells (FOP sample) ~25µg Lane 2: X Lane 3: HB09-1 Lane 4: HB09-8 Lane 5: HB09-9 Lane 6: HB09-11 Lane 7: HB09-20 Lane 8: HB09-20 (6/1/2011)

***Motorcycle man sample p-SMAD HBO9-7 Lysate HBO9-9 Lysate HBO9-19 Lysate HBO9-20 Lysate Normal Shed Cell Lysate Polymorphisms c270 C>T c67-236 delT c67-236 A>a/g c.330+60 G>g/a c642+7 C>c/t c690 G>A 1131 C>c/t c1395+45 G>g/a c1396-93 G>g/a 138 after stop G>A Samples HB09-7 HB09-9 HB08-19 HB08-20*** ***Motorcycle man sample

Lanes 1 2 3 4 5 6 7 8 Mrker Lane 1: Shed Cells (Norm sample) ~20µg Lane 2: HB09-10 Norm Lane 3: HB09-1 Lane 4: HB09-7 Lane 5: HB09-8 Lane 6: HB09-11 Lane 7: HB08-19 Lane 8: HB09-20