Functional Responses in the Additional Cells

1. Functional Responses in the Additional Cells Cell Preparation and Analysis Laboratory and Antibody Laboratory (Dianne DeCamp, Heping Han, Robert Hsueh, Keng-Mean Lin, Yan Ni)

2. The Players

3. Test Ligands

4. Protocols for Calcium Assays in RAW264.7 and J774 Cells RAW264.7 J774 Detachment using PBS/EDTA Adjusting the cell density to 1 x106 cells/ml Loading with Fluo3 AM (3 mM) Recovery Washing Adjusting the cell density to 1 x106 cells/ml stimulate with different ligands Note: Cells are in suspension throughout the calcium assay.

5. 300 250 [Ca2+] (nM) 400 C5a (mM) [Ca2+] (nM) 200 200 300 Lyso- phosphatidic Acid (mM) 400 250 [Ca2+] (nM) [Ca2+] (nM) 200 200 300 400 Platelet-Activating Factor (mM) [Ca2+] (nM) 250 [Ca2+] (nM) 200 200 0 100 200 300 0 100 200 300 Changes in Intracelluar Calcium RAW 264.7 J774A.1 Time (sec) Time (sec)

6. Protocols for cAMP Assays in RAW264.7 Cells RAW264.7 Plate cells overnight in the 96-well plate at 5 x105 cells/ml stimulate with different ligands Stop the reaction by addition of 100% ethanol at different time points Process cell lysate Assay with cAMP Biotrak EIA kit Note: the treatment is performed in the absence of any phosphodiesterase inhibitors.

7. cAMP Responses in RAW 264.7 cAMP Responses in RAW264.7 Cells N=3 Fold Time (min) [ISO] = 1 mM [TER] = 10 mM [PGE] = 50 mM Time (min)

8. Test Ligands

9. Summary/Immediate Future • Initial culture conditions are established for the cells • Previously established protocols and assays are being applied to studies of the additional cell lines. • cAMP and Calcium assays are functional, but can be modified. • Preliminary ligand response testing continues

10. Toll-like Receptor Pathways Lipopolysaccharides Imiquimod. R848 (LPS) Lipid proteins mannans CpG DNA Poly (I:c) Thr10 Oxidized EPA Thr9 Thr3 Thr7 Thr2 Thr6 Oxidized EPA * * * * * * * Cell Mediated Immunity Bacterial death T cells Direct antimicrobial response Influence adaptive Immune response Apoptosis of host cells

11. Multiplex Western Blot with Phosphospecific Antibody Mix 2 RAW 264.7 Cells+/-LPS or IL-4 < P-PKCm < P-STAT3 < P-NFkB p65 > P-JNK <P-IKBa <P-p38MAPK LPS = 10 mg/ml, IL-4 = 0.34 nM

12. IL-4 Pathway

13. Multiplex Western Blot with Phosphospecific Antibody Mix 1RAW 264.7 Cells+/-LPS or IL-4 < P-STAT6 < P-p90RSK < P-Akt > P-ERKs LPS = 10 mg/ml, IL-4 = 0.34 nM

14. 24-hour Serum Deprivation Increases Ligand-Stimulated Protein Phosphorylation in RAW 264.7 Cells • Similar Increases for: • P-STAT6 • P-JNK (long) • P-JNK (short) • P-ERK1 • P-ERK2 • P-p70S6K • P-Ribosomal S6 • Not true for WEHI-231 Small or no Difference: P-Akt, P-NFKB p65, PKC m, IKB a

15. Data from Protein and Cell LabsWestern Blots of Bulk Protein Phosphorylation in RAW 264.7 < 111 < 80 < 61 < 48 < 36 < 25 < 19 < 14 < 6 ____ ____ ____ _____ 60 min 30 min Phospho-threonine and/or phospho-tyrosine antibodies may be useful for future IP and phosphoprotein identification in RAW cells

16. Future • Phosphospecific antibody Mixes 1 and 2 are ready • candidates for monitoring protein phosphorylation • in RAW 264.7 cells • The same is likely true • for or other hematopoietic cells (J774) • for AtT20 (neruorendocrine) cells • based on basal phosphorylation patterns (not shown)

17. The Usual Suspects Cell Preparation and Analysis Laboratory Julie Collins Richard Davis Joella Grossoehme Christine Horvath Jason Polasek Meghdad Rahdar Debalina Siddeeq Megan Smith Melissa Stalder Antibody Laboratory Eduardo Arteaga Becky Fulin Ila Oxendine Greta Sartoni-D’Ambrosia Nick Wong Biren Zhao