Tubes were spun, the nuclear
lysis buffer with proteinase inhibitor was added and sonicated to form shared chromatin of about 200 — 1000 base pairs in length (data not shown).
Lane 1: HEK cells Lane 2: hfRPE cells Lane 3: hfRetina Lane 4: Mouse liver Lane 5: Mouse RPE Lane 6: Mouse retina Lane 7: Zebrafish liver Lane 8: Zebrafish retina PVDF membrane RIPA
lysis buffer with mixed phosphatase and protease inhibitor cocktail
Lane 1: HEK cells Lane 2: hfRPE cells Lane 3: hfRetina PVDF membrane RIPA
lysis buffer with mixed phosphatase and protease inhibitors
Not exact matches
Peripheral blood mononuclear cells were isolated by centrifugation
with 96 % Ficoll (BD), followed by erythrocyte
lysis with ACK
lysis buffer.
At 48 hours, cells were washed twice
with PBS and lysed in 1 × passive
lysis buffer (Dual - Luciferase Reporter Assay, Promega).
Spleens were processed into cell suspensions through a 70 - μm strainer by a syringe plunger in RPMI 1640
with 10 % FBS and then treated
with ACK
lysis buffer to remove RBCs.
Reactions were terminated
with 1 ml of ice - cold PBS, and cells were centrifuged and resuspended in 50 μl of cell
lysis buffer containing 1 % NP - 40 and protease inhibitors.
Beads were washed by centrifugation at 100g, 4 °C for 2 min
with lysis buffer containing 2 mM Na3VO4 and boiled
with 2 × protein sample
buffer at 95 °C for 10 min.
Spleens were then treated
with ACK
lysis buffer to remove RBCs.
After extensive washing
with immunoprecipitation
lysis buffer, the immunoprecipitated proteins were analyzed by immunoblotting using specific antibodies against DDX3 and FLAG epitope (ab1257, Abcam, Cambridge, United Kingdom).
For the coimmunoprecipitation experiments, HuH - 7 cells transfected
with pcDNA - SRα / FLAG - Sp 1 were harvested and cell lysates were prepared using immunoprecipitation
lysis buffer [20 mmol / L Tris - Cl (pH 7.5), 150 mmol / L NaCl, 10 % glycerol, and 1 % Triton X-100].
NHLF cells transfected
with siRNA molecules were lysed in High Salt ELB
lysis buffer [1 M Tris pH 8.0, 1 % NP - 40, 250 mM NaCl, 5 mM EDTA] supplemented
with protease and phosphatase inhibitors (1x G - Biosciences Protease Arrest, 200 µM Na3VO4, and 1 mM PMSF).
Cells were lysed
with TN1
lysis buffer (50 mM Tris, 125 mM NaCl, 1 % Triton X-100, 10 mM EDTA, 10 mM sodium fluoride and 10 mM sodium pyrophosphate) supplemented
with protease inhibitor cocktail (1.2 mM AEBSF, 0.46 μM aprotinin, 14 μM bestatin, 12.3 μM E-64, 112 μM leupeptin, 1.16 μM pepstatin)(Amresco; Solon, OH)-RRB- and 1 mM sodium orthovanadate (Enzo Life Sciences; Farmingdale, NY).
For this protocol, samples were lysed in 1 × RIPK2
lysis buffer [50 mM Tris (pH 7.5), 0 mM MgCl2, 1 % Triton X-100, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA,
with freshly added 1 mM β - glycerophosphate and protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin)-RSB- and immunoprecipitated overnight using 1.5 μg rabbit anti-RIPK2 antibody.
Hippocampus or frontal cortex samples were homogenized in
lysis buffer (10 mm Tris, pH 7.5, 10 mm NaCl, 3 mm MgCl2, 1 mm EDTA, and 0.05 % NP - 40)
with protease inhibitors (Halt protease inhibitor cocktail, Thermo Scientific) and protein concentration was determined by Bradford assay (Coomassie Plus, Thermo Scientific).
48 hours after transfection, cells were lysed
with 100 µL / well 1 × passive
lysis buffer (PLB, Promega) for 15 minutes
with shaking.
Confluent 10 cm plates were fixed
with 1 % PFA at room temperature for 10 minutes, lysed in SDS
lysis buffer (50 mM Tris, 10 mM EDTA, 1 % SDS, Roche Complete protease inhibitors, pH 8.1), scraped and collected into 1.5 mL microcentrifuge tubes, and DNA was sonicated to 200 — 800 bp fragments
with a Branson Sonifier 250 set to 30 % power / 90 % duty, four 10 second pulses.