Mouse anti ha antibody coupled agarose beads

HEK 293T cells were transfected with plasmids directing the expression of HA-tagged WT or mutant L13a proteins. Twenty-four hours post-transfection, cells were lysed and 100 µg of lysates were immunoprecipitated with mouse anti-HA antibody coupled agarose beads (Sigma) or nonantibody-couple beads as a control in the buffer 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl₂, 0.05% Triton X-100, 50 mM HEPES (pH 7.5). Ten micrograms of lysates before immunoprecipitation were used as an input for nucleolin. Total protein from the immunoprecipitates was subjected to SDS-PAGE, followed by immunoblot analysis with mouse monoclonal anti-nucleolin antibody (Millipore, Catalog# MABC587). To confirm the equal efficiency of immunoprecipitation in all of the samples immunoblot was also performed with rabbit anti-HA antibody (Abcam).

HEK 293T cells were transfected with plasmids directing the expression of HA-tagged WT or mutant L13a proteins. Twenty-four hours post-transfection, cell lysates were prepared and subjected to immunoprecipitation using mouse anti-HA antibody coupled agarose beads (Sigma) or nonantibody-coupled beads as a control in the buffer 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl₂, 0.05% Triton X-100, 50 mM HEPES (pH 7.5) (Mazumder et al. 2003 (link)). Total L13a-bound RNA was extracted from the immunoprecipitates using TRIzol. The extracted RNA was reverse-transcribed using random hexamers and reverse transcriptase (superscript) following the manufacturer's protocol (Invitrogen). The synthesized first-strand cDNA was subjected to reverse transcription-PCR (RT-PCR) using human 28S rRNA specific primer pairs. Forward primer sequence 5′-GAAGTTTCCCTCAGGATAGCT-3′ and reverse primer sequence 5′-GCAGGTGAGTTGTTACACACT-3′ were used. The PCR product of 355 base pairs was analyzed by agarose gel electrophoresis. Immunoblotting with anti-HA antibody was used to confirm the presence of L13a in the immunoprecipitate.