Proxima c16 phi imaging system

Total RNA and cDNA were prepared respectively by the RNeasy Mini Kit (Cat#74104, QIAGEN) and the RevertAid First Strand cDNA Synthesis Kit (Cat#K1622, Thermo Scientific). The synthesized cDNA was used as a template for PCR amplification of PIP5K1A (Forward primer: AGA AGA TTC CCT GCG TTC ACC, Reverse primer: GAT CTA GAC TAT GGG TGA ACT CTG ACT CTG) and GAPDH (Forward primer: AAC AGC GAC ACC CAC TCC TC, Reverse primer: GGA GGG GAG ATT CAG TGT GGT) by using the Phire Hot Start II DNA Polymerase Kit (Cat#F122S, Thermo Scientific). The PCR product was analyzed by gel electrophoresis in 1% agarose. The signal was captured and documented with the Proxima C16 Phi+ imaging system (Isogen Lifescience). Densitometric quantification was performed by the software ImageJ 1.50i Software (NIH, Baltimore, MD, Unites States) and represented as fold change relative to control and was normalized relative to GAPDH.

Subcellular fractionation, immunoprecipitation analysis, and immunoblotting were performed as described previously (Semenas et al., 2014 (link)). Briefly, protein from different subcellular fractions was prepared by using Subcellular Protein Fractionation Kit for Cultured Cells (Cat#78840, Thermo Scientific™) according to the manufacturer’s protocol. For immunoprecipitation analysis, the Protein G Sepharose™ 4 Fast Flow beads (Cat#17-0618-01, GE Healthcare) and anti-PIP5K1α antibody (Cat#15713-1-AP, Proteintech) were applied to pull down PIP5K1a from the protein lysates. The rabbit IgG isotype (Cat#02-6102, Invitrogen) was used as a control. For immunoblotting, antibodies against β-tubulin (Cat#075K4875, Sigma-Aldrich) and Lamin B (Cat#sc-6216, Santa Cruz) were used, respectively, as controls for cytoplasmic and nuclear fractions. The signal was captured and documented with the Proxima C16 Phi+ imaging system (Isogen Lifescience). Densitometric quantification of immunoblots was performed by the software ImageJ 1.50i Software (NIH, Baltimore, MD, Unites States) and represented as fold change relative to control and was normalized relative to actin or GAPDH bands.