Pharmacological approach showing the involvement of S-palmitoylation in NLD targeting the endo-PM in Arabidopsis pollen. (A–D) Confocal imaging of mature pollen expressing NLD_full:mCitrine after 2-h treatment with mock (EtOH; A) or with 150 µM 2-BP (B–D). After mock treatment, NLD_full:mCitrine mainly localizes at the endo-PM (A), whereas 2-BP treatment leads to three different types of localization: endo-PM (B), noncontinuous (“spotty”) fluorescence signal at endo-PM (C), and low fluorescent signal in cytosol (D). Scale bars = 20 µm. (E) Quantification of drug treatment effect on NLD subcellular localization by counting the three different classes of localization patterns after 2-BP treatment. Data were obtained from five independent experiments (mean ± SD). ***, significant difference as compared with the mock treatment (P < 0.001; χ² test, n = total number of counted mature pollens). (F and G) Verification of transgene expression for the three protein fusions harboring mutation on C10 or/and C423 residues (NLD_C10S:mCitrine, NLD_C423S:mCitrine, and NLD_C10S,C423S:mCitrine; G) together with the two fusions (NLD_full:mCitrine and NLD_trunc:mCitrine; F). RT-PCR was done on one or two selected lines for each construct, and three independent biological replicates were used per line. Minus RT samples were RT reactions conducted without RT enzyme to evaluate possible gDNA contaminations in RNA extractions.