Ab38007

RNA immunoprecipitation (RIP) analysis from whole cell extracts was performed as previously described (Min et al., 2017 (link)). Briefly, cells were lysed in buffer containing 20 mM Tris-HCl (pH7.5), 100 mM KCl, 5 mM MgCl₂, 0.5% NP-40 and cleared by centrifugation. The lysates were incubated with protein A-Sepharose beads coated with antibodies against PGK1 (ab38007, Abcam), PGAM1 (ab96622), ENO1 (ab155102), GAPDH (2275-PC-100, R&D Systems), Pinin (18266, Proteintech; ab244250) or nucleolin (ab22758) with control IgG (Sigma) for 1 h at 4°C. After the beads were washed four times with NT2 buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl₂, 0.05% NP-40], the immunoprecipitates were treated with 20 units of RNase-free DNase I for 15 min at 37°C and proteinase K (0.5 mg/ml) in 0.1% SDS for 15 min at 55 °C to remove DNAs and proteins, respectively. The RNAs isolated from the IPs by acidic phenol extraction were then subjected to RT-qPCR using the primers listed in Table S1. The RIP results were normalized to 18S rRNA or GAPDH mRNA.