PRL1 associates with the Pol II and DCL1 complexes.

<p>(A) and (B) Co-immunoprecipitation (Co-IP) between PRL1 and Pol II. Protein extracts from transgenic plants containing PRL1-YFP were incubated with Anti-YFP or anti-RPB2 antibodies to precipitate PRL1-YFP or Pol II. PRL1-YFP and RBP2 were detected with western blot and labeled on the left side of the picture. Ten percent of input proteins were used for IP and one percent of input proteins were used for Co-IP. (C) BiFC analysis of PRL1 with DCL1, HYL1, SE, AGO1 and CDC5. Paired cCFP- and nVenus-fusion proteins were co-infiltrated into <i>N. benthamiana</i> leaves. The BiFC signal (Yellow fluorescence) was detected at 48 h after infiltration by confocal microscopy, assigned as green color and marked with arrow. 30 nuclei were examined for each pair and an image is shown. Red: auto fluorescence of chlorophyll. (D) and (E) Co-immunoprecipitation between PRL1 and DCL1. (F) and (G) Co-immunoprecipitation between PRL1 and SE. PRL1-YFP or YFP were co-expressed with DCL1-MYC and SE-MYC in <i>N. benthamiana</i>, respectively. Anti-YFP and anti-MYC (MBL) antibodies were used to detect YFP- and MYC-fused proteins, respectively. The protein pairs in the protein extracts were indicated on the on tope of the picture and proteins detected by western blot were indicated on the left side of the picture. Ten percent of input proteins were used for IP and one percent of inputs proteins were used for Co-IP.</p

PRL1 and CDC5 synergistically regulate miRNA accumulation.

<p>(A) Morphological phenotypes of Col, <i>cdc5-1</i>, <i>prl1-2</i> and <i>cdc5-1 prl1-2</i>. (B) The abundance of miRNAs is lower in <i>cdc5-1 prl1-2</i> than that in <i>cdc5-1</i> or <i>prl1-2</i>. Small RNAs were detected by Northern Blot. To determine the amount of miRNAs, radioactive signals of miRNAs were normalized to U6 RNA. The number represents the relative abundance compared to Col (set as 1) quantified by three repeats (P<0.05). (C) The abundance of pri-miRNAs is reduced in <i>cdc5-1 prl1-2</i>. The levels of pri-miRNAs in various mutants were determined by qRT-PCR, normalized to <i>UBQUITIN5</i> (<i>UBQ5</i>) and compared with those of Col (set as 1). Standard deviation of three technical replications was shown as error bars. **: P<0.01. (D) miR162b production from <i>pre-miR162b</i> in Col, <i>cdc5-1 prl1-2</i>, <i>cdc5-1</i> and <i>prl1-2</i>. The reaction was stopped at 120 min. The radioactive signals of miR162b were normalized to input. The number represents the relative production in various genotypes compared to Col (set as 1) quantified by three repeats (P<0.05).</p

PRL1 is required for miRNA maturation <i>in vitro</i>.

<p>(A) and (B) A schematic diagram of the <i>MIR162b</i> (A) and <i>pre-miR162b</i> (B) used <i>in vitro</i> processing assay. (C) and (D) The amount of miR162b produced from <i>MIR162b</i> and <i>pre-miR162b</i> were reduced in <i>prl1-2</i>. Proteins were isolated from inflorescences of <i>prl1-2</i> and Col and incubated with <i>MIR162b</i> or <i>pre-miR162b</i>. The reactions were stopped at various time points as indicated in the picture. (E) and (F) Quantification of miR162b production in <i>prl1-2</i> compared to that in Col. Quantification analysis was performed at 80 min. The radioactive signal of miR162 were normalized to input and compared with that of Col. The amount of miR162 produced in Col was set as 1. The value represents mean of three repeats (*** <i>P</i><0.001; t-test).</p

The role of PRL1 in siRNA biogenesis.

<p>(A) PRL1 interacts with DCL3 and DCL4. Co-IP was performed to detect the interaction of PRL1 with DCL3 or DCL4. MBP and MBP-PRL1 fused protein were expressed in <i>E.coli</i>. YFP, DCL3-YFP and DCL4-YFP were expressed in <i>N. benthamiana</i> leaves. Anti-YFP was used for IP. For loading, ten percent and one percent of input proteins were used for IP and Co-IP, respectively. (B) <i>prl1-2</i> impairs siRNA production from double-stranded RNAs (dsRNAs). Protein extracts isolated from inflorescences of Col, <i>prl1-2</i> and <i>prl1-2</i> containing a PRL1-YFP transgene were incubated dsRNAs for 120 min. dsRNAs were synthesized through <i>in vitro</i> transcription of a DNA fragment (5′ portion of <i>UBQ5</i> gene, ∼460 bp) under the presence of [α-³²P] UTP.</p

MS/MS spectrum of multiple esterified glutamic acid residues.

(A) Two MS/MS spectra of HC 378–399 with identical m/z showing the esterification at E387 and E389 differentiated by distinct y11+ and y12+ ions. (B) MS/MS spectra of tryptic peptide HC 300–308 showing esterification at either E300 or E301. The highest intensity singly charged ion at m/z 1556.65 is a MS/MS fragment of a peptide containing the N-acetylglucosamine remnant of a liberated N- glycan. Color code: blue square-N-acetylglucosamine; red triangle-fucose; green circle-mannose. (C) MS/MS spectrum of HC 282–295 demonstrates E290 as the site of esterification.</p

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General scheme of the esterification reaction of an acid with a sugar alcohol.

General scheme of the esterification reaction of an acid with a sugar alcohol.</p

Total Ion Chromatogram (TIC) and MS/MS spectra showing the site- specific identification of esterified glutamic acid residues within the tryptic peptide HC 263–281.

(A) Total Ion Chromatogram showing distinct esterification peaks with identical m/z eluting prior to the unmodified peptide peak. (B) MS/MS spectrum of E279 esterification within the tryptic peptide HC 263–281. Despite sharing an identical parent mass, this peptide possesses a distinct y4+ -y5+ ion series compared to the E276 esterified residue in HC 263–281 (Fig 2B).</p

Summary of observed esterified glutamic acid residues, location and percent esterification.

Summary of observed esterified glutamic acid residues, location and percent esterification.</p