I. Pyrophosphate Assays

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I. Pyrophosphate Assays Research Update KL 8/17/2010

Pyrophosphate Concentration Day 0-2 Functional ANK positively regulates osteoblast development (APase, BSP, osteocalcin, osterix, runx2, and type I collagen) (Kirsch, JBMR, 2010)

II. ANK siRNA KD: Time point and Dosage

ANK (Santa Cruz) 8/17/2010 250 kDa ANK( 54kDA) 150 kDa 200mM Day 4 ANK 40mM Day 4 ANK 200mM Day 2 ANK 200mM Day 3 ANK 40mM Day 3 ANK Scramble #2 200mM Day 3 40mM Day 2 ANK MC3T3 Ladder 250 kDa ANK( 54kDA) Blocking Solution: 5% NFM, 1hr RT Primary: 1:200 Rabbit Polyclonal Anti-ANK, 1hr RT Secondary: 1:4000 Goat Anti-Rabbit, 1hr RT Chemiluminescence, 150 kDa 100 kDa 75 kDa 50 kDa Actin( 42kDA) Blocking Solution: 5% BSA, 1hr RT Primary: 1:1000 Anti-Actin IgG2A, overnight, 4C Secondary: 1:10,000 Goat Anti-Mouse Ig-HRP, 1hr RT Chemiluminescence, sec exposure 37 kDa 25 kDa 20 kDa 15 kDa

ANK (Santa Cruz) 8/17/2010 ANK( 54kDA) 250 kDa Scramble #2 40nM Day 3 Scramble 40nM Day 4 Scramble 200nM Day 3 Scramble 200nM Day 4 Scramble #2 40nM Day 2 Scramble #2 40nM Day 4 Scramble 40nM Day 2 Scramble 40nM Day 3 Scramble 200nM Day 2 Ladder ANK( 54kDA) Blocking Solution: 5% NFM, 1hr RT Primary: 1:200 Rabbit Polyclonal Anti-ANK, 1hr RT Secondary: 1:4000 Goat Anti-Rabbit, 1hr RT Chemiluminescence, 250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa Actin( 42kDA) Blocking Solution: 5% BSA, 1hr RT Primary: 1:1000 Anti-Actin IgG2A, overnight, 4C Secondary: 1:10,000 Goat Anti-Mouse Ig-HRP, 1hr RT Chemiluminescence, sec exposure 25 kDa 20 kDa 15 kDa 10 kDa

III. Western blot: TRPV4 and IFT88 siRNA Knockdowns (NB110-74960 and H00008100-A01)

IFT88 (Novus) 8/16/2010 Polaris 2200pmol Scrambled 600pmol Negative Vector Control Polaris 600pmol MC3T3 Ladder IFT88( 95kDA) Blocking Solution: 5% NFM, 1hr RT Primary: 1:500 Mouse Anti-IFT88, overnight, 4C Secondary: 1:2000 Goat Anti-Mouse, 1hr RT Chemiluminescence, 15 sec exposure 250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa Actin( 42kDA) Blocking Solution: 5% BSA, 1hr RT Primary: 1:1000 Anti-Actin IgG2A, overnight, 4C Secondary: 1:10,000 Goat Anti-Mouse Ig-HRP, 1hr RT Chemiluminescence, 30 sec exposure 25 kDa 20 kDa 15 kDa 10 kDa

TTC10 (Abcam) 7/14/2010 Scrambled 2200pmol Polaris 600pmol WT MC3T3 Ladder 250 kDa 150 kDa 100 kDa 75 kDa TTC10( 95kDA) Blocking Solution: 1% BSA, 1hr RT Primary: 1:1000 Goat Polyclonal Anti-YTC10, overnight, 4C Secondary: 1:5000 Rabbit Polyclonal Anti-Goat, 1hr RT Chemiluminescence, 5 sec exposure 50 kDa Actin( 42kDA) Blocking Solution: 5% BSA, 1hr RT Primary: 1:1000 Anti-Actin IgG2A, overnight, 4C Secondary: 1:10,000 Goat Anti-Mouse Ig-HRP, 1hr RT Chemiluminescence, 30 sec exposure 37 kDa

TRPV4 (Novus) 8/16/2010 TRPV4_1 600pmol TRPV4_1 2200pmol Scrambled 600pmol Scrambled 2200pmol Scrambled #2 600pmol Scrambled #2 2200pmol MC3T3 Ladder TRPV4( 98kDA) Blocking Solution: 5% NFM, 1hr RT Primary: 1:1000 Rabbit Polyclonal Anti-TRPV4, O/N, 4C Secondary: 1:4000 Goat Anti-Rabbit, 1hr RT Chemiluminescence, 30 sec exposure 250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa Actin( 42kDA) Blocking Solution: 5% BSA, 1hr RT Primary: 1:1000 Anti-Actin IgG2A, overnight, 4C Secondary: 1:10,000 Goat Anti-Mouse Ig-HRP, 1hr RT Chemiluminescence, 30 sec exposure 25 kDa 20 kDa 15 kDa 10 kDa

TRPV4 (Alomone) 7/14/2010 TRPV4_1 600pmol TRPV4_1 2200pmol TRPV4_2 Scrambled 600pmol Scrambled 2200pmol TRPV4_2 2200pmol Ladder WT MC3T3 250 kDa TRPV4 (98kDa) Blocking Solution: 1% BSA TBS-Tween, 1 hr RT Primary: 1:200 Rabbit PolyclonalAnti-TRPV4 O/N 4C Secondary: 1:4000 Goat Polyclonal Anti-Rabbit HRP, 1hr RT 150 kDa 100 kDa 75 kDa Chemiluminescent Detection FUJI Mini 4000: Standard, 30 sec exposure 50 kDa 37 kDa Actin( 42kDA) Blocking Solution: 5% BSA, 1hr RT Primary: 1:1000 Anti-Actin IgG2A, overnight, 4C Secondary: 1:10,000 Goat Anti-Mouse Ig-HRP, 1hr RT Chemiluminescence, 30 sec exposure

TRPV4 Literature TRPV4 localizes to primary cilia and transports Ca2+ Cholangiocytes (LaRusso) OsmosensitiveCa2+ increases in a TRPV4-, ciliary-, and extracellular Ca-dependent manner Porcine Articular Chondrocytes (Guilak) Block TRPV4 (GSK205 antagonist)reduced Ca2+ responsiveness and PGE2 production Removal of primary cilia eliminated Ca2+ signaling

TRPV4 Literature Bone (Carmeliet) TRPV4 knockout increases bone mass by impairing bone resorption Intracellular Ca2+ signaling is required for osteoclast maturation and differentiation (TRAP staining, NFATc1, NFATc1-dependent genes :c-fos, c-jun, calcineurin mRNA) Trvp4(-/-) mice had increased bone mass Osteoblast #, Osteocalcin, Runx2, alkaline phosphatase were comparable between Trvp4(-/-) and WT, TRPV4 is NOT crucial for osteblast proliferation or differentiation TRAP:staining of tartrate-resistant acid phosphatase in osteoclasts C-fos: key regulator of osteoclast-macrophage lineage determination and bone remodeling NFATc1:master regulator of nuclear factor kappaB ligand-induced osteoclast differentiation C-jun: RANKL-activated transcription factor that regulates osteoclastogenesis Calcineurin: necessary downstream mediator for osteoclast differentiation

Knockdown TRPV4 again (Lipo or Neon?) Next: MC3T3s: Knockdown TRPV4 again (Lipo or Neon?) Find positive lysate Run another Western and PCR Run flow Measure Ca2+? COX2, PGE2