Benzamidine hydrochloride

HEK293T cells were harvested and resuspended in NET-2 buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), 2 mM benzamidine hydrochloride (Sigma-Aldrich), 0.05% NP-40 (IGEPAL CA-630; Sigma-Aldrich), 10 mM sodium fluoride (Sigma-Aldrich), and 0.25 mM sodium orthovanadate (Sigma-Aldrich)]. The suspended cells were sonicated and pre-cleared with protein A or G agarose 4 B beads (Incospharm) for 1 h at 4 °C. The pre-cleared samples were incubated with FLAG M2 affinity gel (Sigma-Aldrich) or antibody-conjugated beads for 3 h at 4 °C. The beads were washed four times with NET-2 buffer, and the bead-bound proteins were eluted using 2× sample buffer. The samples were analyzed by western blotting.
Where indicated, cell extracts before IP were treated with RNase A. Total RNAs were purified with TRIzol Reagent (Invitrogen). Then, purified total RNA samples were mixed with in vitro-transcribed FLuc RNAs as a spike-in. The amounts of mRNAs were quantified using quantitative reverse-transcription PCR (qRT-PCR), and the level of endogenous GAPDH mRNAs were normalized to FLuc RNAs. 5′-TGGCAAATTCCATGGCACC-3′ (sense) and 5′-AGAGATGATGACCCTTTTG-3′ (antisense) oligonucleotides for amplification of GAPDH mRNAs and 5′-CAACACCCCAACATCTTCG-3′ (sense) and 5′-CTTTCCGCCCTTCTTGGCC-3′ (antisense) oligonucleotides for amplification of FLuc RNAs.

HEK293T cells were harvested and lysed using NET-2 buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), 2 mM benzamidine hydrochloride (Sigma-Aldrich), 0.05% NP-40 (IGEPAL^® CA-630; Sigma-Aldrich), 10 mM sodium fluoride (Sigma-Aldrich), and 0.25 mM sodium orthovanadate (Sigma-Aldrich)]. The cell extracts were sonicated and precleared with protein G agarose 4B beads (Incospharm) for 1 h at 4 °C. Next, the samples were incubated with either antibody-conjugated beads or FLAG M2 affinity gel (Sigma-Aldrich) at 4 °C. After 3 h, the beads were washed four times with NET-2 buffer and eluted with 2× sample buffer. The samples were analyzed using western blotting.

Calcium ionophore (A23187), benzamidine hydrochloride, N-acetyl-Leu-Glu-His-Asp trifluoro methylcoumarin (AC-LEHD-FMC), acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (AC-DEVD-AMC), sodium orthovanadate, dimethyl sulfoxide (DMSO), fluorescein isothiocyanate (FITC)-labeled annexin V, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester (CM-H2DCFDA), CHAPS, rhodamine 123, leupeptin hydrochoride, N-(2-Hydroxyethyl) piperazine-N′-ethanesulfonic acid (HEPES), fura-2/AM, monoclonal anti-phosphotyrosine antibody, acridine orange 10-nonyl bromide (NAO) and dithiothreitol (DTT) were from Sigma Chemicals, St. Louis (USA). Monoclonal anti-cytochrome c antibody and anti-β-actin were from Epitomics Burlingame, CA (USA). Anti-Caspase-3 antibody was from Santa Cruz Biotechnology, Inc. Texas (USA). Collagen type-I was from Chrono-log Corporation, Pennsylvania (USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1, 1-diphenyl-2-picrylhdrazyl (DPPH) were from HiMedia Laboratories, Mumbai (India). Lactate dehydrogenase (LDH) kit was from AGAPPE diagnostics Ltd., Kerala (India). γ-glutamyl p-nitroanilide and glycylglycine were from Sisco Research laboratories Pvt Ltd., Mumbai (India). All other reagents were of analytical grade.