Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 1. ICAM-1 cross-linking in- duced time-dependent activation of SRC tyrosine kinases. TNF--pre- treated ECs were incubated for 30 min with 10 g/ml mouse anti-human ICAM-1 antibody and washed. A cross-linking sec- ondary antibody was added for 0–15 min, and the activity of SRC was evaluated by an in vitro kinase assay using Sam68- (331–443) as a substrate after immuno- precipitating SRC. Tyrosine phosphoryla- tion of Sam68-(331–443) was detected by immunoblot using an anti-phosphoty- rosine antibody as described under “Ex- perimental Procedures.” A, representa- tive immunoblot showing activation of SRC tyrosine kinases in response to ICAM-1 cross-linking. The amount of im- munoprecipitated SRC was also deter- mined for each sample. Lanes 1–5, SRC activity in ECs prior to (lane 1) or 0.25–15 min after ICAM-1 cross-linking (lanes 2–5). B, densitometric analysis of immu- noblots. The activity of SRC tyrosine ki- nases was normalized by the amount of immunoprecipitated SRC. Data are pre- sented as fold changes over the non-cross- linked controls and expressed as means S.E. (n 4). *, p 0.05 when compared with controls.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Activity Assay, In Vitro, Kinase Assay, Western Blot, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 2. Activation of SRC tyrosine ki- nases was inhibited by allopurinol, a xanthine oxidase inhibitor (A), as well as Me2SO, a hydroxyl radical scav- enger, and deferoxamine, an iron che- lator (B). ECs were treated with 10 g/ml anti-ICAM-1 along with 0.3 mg/ml allo- purinol, 1% Me2SO, or 1.5 mM deferoxa- mine, or their respective control vehicle for 30 min and washed. A cross-linking sec- ondary antibody was added for 0–6 min, and SRC activity was evaluated as de- scribed under “Experimental Procedures.” Open bars, no cross-linking; closed bars, cross-linking for 6 min. Data are expressed as fold changes from the non-cross-linked controls in vehicle-pretreated samples and presented as mean S.E. (n 4). *, p 0.05 when compared with the non-cross- linked controls; #, p 0.05 when compared with the vehicle-pretreated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Control, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 3. Modulation of SRC activity by PAO, a tyrosine phosphatase in- hibitor. ECs were treated with 10 g/ml anti-ICAM-1 along with control vehicle or 20 M PAO for 30 min and washed. A cross-linking secondary antibody was added for 0–6 min, and SRC activity was evaluated as described under “Experi- mental Procedures.” Data are expressed as fold changes from the non-cross-linked controls in vehicle-pretreated samples, and presented as mean S.E. (n 4). *, p 0.05 when compared with the non- cross-linked controls; #, p 0.05 when compared with the vehicle-pretreated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Control

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 4. Activation of SRC tyrosine kinases required SHP-2. ECs were treated with 10 nM control or SHP-2 an- tisense oligonucleotides as described un- der “Experimental Procedures.” A, the ef- fect of SHP-2 antisense on the protein expression of SHP-2 or SHP-1 in ECs as examined by immunoblot. B, the effect of SHP-2 antisense on SRC activity before or after ICAM-1 cross-linking for 6 min. Data are expressed as fold changes from the non-cross-linked controls and pre- sented as means S.E. (n 8). *, p 0.05 when compared with the non-cross- linked controls; #, p 0.05 when com- pared with the control antisense-treated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Control, Expressing, Western Blot, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 5. Immunoprecipitated SHP-2 from ECs can dephosphorylate the phospho-SRC peptide at residue Tyr-530. SHP-2 was immunoprecipitated (IP) from ECs, and dephosphorylation of the phospho-SRC peptide at residue Tyr-530 was examined as described under “Experimental Procedures.” A, examples of two independent samples showing that immunoprecipitated SHP-2 can decrease the phosphorylation levels of the phospho-SRC peptide. Top gel, SHP-2 was specifically immunoprecipitated using a SHP-2 antibody. Bottom gel, incubation with the immunoprecipitated SHP-2 resulted in a decrease in the phosphorylation levels of the phospho-SRC peptide. B, densitometric analysis of the decrease in the phosphorylation levels of the phospho-SRC peptide as shown in A. Data are expressed relative to the control samples and presented as means S.E. (n 5). *, p 0.05 when compared with control samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Residue, De-Phosphorylation Assay, Phospho-proteomics, Incubation, Control

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 6. Activation of p38 MAPK in- duced by ICAM-1 cross-linking was inhibited by PP2, an inhibitor of SRC tyrosine kinases. ECs were incubated with 10 g/ml anti-ICAM-1 antibody along with vehicle or 20 M PP2 for 30 min and washed. The cells were either left untreated or treated with cross-linking secondary an- tibody for 6 min. The activity of p38 MAPK was evaluated by an in vitro kinase assay using ATF-2 as a substrate. A, activity of p38 MAPK as evaluated by phosphorylation of ATF-2. As a loading control, the amount of ezrin in the samples used for immunopre- cipitation was also examined. B, densitomet- ric analysis of ATF-2 phosphorylation as in A. Open bars, no cross-linking; closed bars, cross-linking ICAM-1 for 6 min. The data are expressed as fold changes from the non- cross-linked controls in vehicle-pretreated samples and are presented as means S.E. (n 6 or 7). *, p 0.05 when compared with the non-cross-linked controls.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Activity Assay, In Vitro, Kinase Assay, Phospho-proteomics, Control

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: bioRxiv

Article Title: Human hepatocyte PNPLA3 148M exacerbates rapid non-alcoholic steatohepatitis development in chimeric mice

doi: 10.1101/2020.11.19.387613

Figure Lengend Snippet: a) ALT and AST activity in serum from huFNRG mice on chow and after 4 to 12 weeks on WD. Symbols individual mice, bars are median, unpaired t -test **p<0.005, ****p<0.0001. b) H&E staining from a huFNRG mouse liver after 8 weeks on WD. Black arrow indicates hepatocyte ballooning degeneration and white arrow indicates lobular inflammation. c) Staining against human nuclear mitotic apparatus-1 in the liver of a huFNRG mouse after 4 weeks on WD. Black arrow indicates hepatocyte ballooning. d) Fractions of huFNRG mice with human hepatocyte ballooning degeneration on chow and WD over time. Number of mice with ballooning per group at bottom of bars. e) Fractions of huFNRG mice with human hepatocyte ballooning degeneration by grade of steatosis. Number of mice with ballooning per group at bottom of bars. f) NAFLD activity score (NAS) in the human graft of huFNRG mice on chow and after 4 to 20 weeks on WD. Symbols individual mice, bars are median, unpaired t -test **p<0.005, ****p<0.0001. g) Picrosirius Red staining for collagen in livers from huFNRG mice on chow and after 4 to 20 weeks on WD. h) Fibrosis stages in the human graft of huFNRG liver on chow and after 4 to 20 weeks on WD. Mouse numbers at bottom of bars.

Article Snippet: 2009, Chicago: ASCP Press., 114-123], and with Picrosirius Red Stain (PSR) (Abcam cat#ab150681) and Masson's Trichrome (Newcomer Supply cat#9179A, USA) in accordance with manufacturer’s supplied procedures.

Techniques: Activity Assay, Staining

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 depletion leads to cytoplasmic accumulation of endogenous 5′SS motif containing mRNAs (or intronic polyadenylated transcripts). ( A ) Workflow for RNA Frac-seq. ZFC3H1- or control-depleted U2OS cells were fractionated into nuclear and cytoplasmic/ER fractions (see Materials and Methods for more details). RNA was purified from these fractions and from total cell lysates, and then analyzed by Illumina sequencing. ( B ) Nuclear “N” and cytoplasmic “C” fractions were collected from ZFC3H1- or control-depleted U2OS cells, then separated by SDS-PAGE and analyzed by immunoblot for nuclear (Aly), ER (Trap-α), and cytoplasmic (tubulin) protein markers. ( C ) Lysates collected from ZFC3H1- or control-depleted U2OS cells (96 h post-transduction with lentiviral-delivered ZFC3H1-2 shRNA) were analyzed by immunoblot for ZFC3H1 and tubulin. ( D ) Fold change in total levels of intronic polyadenylated (IPA) transcripts (ZFC3H1 depletion vs. control depletion) ( x -axis), plotted against the change in the total levels of fully processed mRNA (using cUTR reads, y -axis). Each dot corresponds to reads from one gene that is known to produce IPA transcripts (listed in Supplemental Table 1 ). Note that ZFC3H1 depletion leads to the up-regulation of IPA transcripts, but not fully processed mRNAs. ( E ) ( Top ) Schematic of a fully processed mRNA and IPA transcript generated from the same gene. Note that the IPA transcript is generated from a 3′ cleavage/polyadenylation signal in the first intron and contains a 5′SS motif. ( Bottom ) genome browser tracks of the PCF11 gene in control- “shCon” or ZFC3H1-depleted “shZFC” cells at 500× and 50× resolution. Note the large peak for the IPA transcript (intronic cleavage/polyadenylation site is denoted with a red arrow), which is up-regulated in ZFC3H1-depleted cells. Also note that the reads corresponding to the full-length transcript are unaffected by ZFC3H1 depletion. ( F ) Similar to D , except that the fold change of IPA transcript levels in the cytoplasmic fraction (ZFC3H1 depletion vs. control depletion, x -axis) is plotted against the fold change in the nuclear fraction (ZFC3H1 depletion vs. control depletion, y -axis). Note that ZFC3H1 depletion leads to cytoplasmic accumulation of many IPA transcripts (compare blue dots to red). To account for reads from fully processed mRNAs, the IPA transcript levels are normalized to the cUTR transcript levels of the same gene. ( G ) Similar to E , except the IPA peaks in the nuclear and cytoplasmic fractions are shown for PCF11 . In control-depleted cells, reads from the PCF11 IPA are enriched in the nuclear, but not the cytoplasmic fractions. In ZFC3H1-depleted cells, reads from the PCF11 IPA are at comparable levels in the nuclear and cytoplasmic fractions. ( H – J ) Genome browser tracks of three IPA transcript-producing genes, “ CCDC71 ,” “ BRD3 ,” and “ ZFPM1 .” Note the accumulation of IPA transcripts in the cytoplasmic fractions upon ZFC3H1 depletion. The intronic 3′ cleavage/polyadenylation sites are denoted by red arrows.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, Purification, Illumina Sequencing, SDS Page, Western Blot, Transduction, shRNA, Generated

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 is required for the nuclear retention of 5′SS motif containing mRNAs. ( A ) U2OS cells were treated with different lentivirus shRNAs against ZFC3H1 (“ZFC3H1-1” and “ZFC3H1-2”), MTR4 (“MTR4-1 + 2”) or control shRNA. Lysates were collected 96 h post-transduction, separated by SDS-PAGE and immunoprobed for ZFC3H1, MTR4, or tubulin. Note that to effectively deplete MTR4, cells were treated with lentivirus containing two shRNA plasmids. ( B ) Schematic of the intronless ( Δi ) ftz reporter ( ftz-Δi ) construct used in this study, with and without the V5-His element in the 3′UTR ( ftz-Δi-5 ′ SS ). Note that the V5-His element contains a consensus 5′SS motif, which promotes nuclear retention. ( C , D ) Control-, MTR4-, and ZFC3H1-depleted cells were transfected with the intronless ftz reporter plasmid (± 5 ′ SS ). Eighteen to twenty-four hours later, the cells were fixed and the mRNA was visualized by FISH. Note that depletion of ZFC3H1, but not MTR4, caused the cytoplasmic accumulation of the ftz-Δi-5′SS mRNA. Representative images are shown in C (scale bar, 10 µm) and quantification is shown in D . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for D. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( E ) Schematic of CCDC71 -IPA reporter used in this study (see also H). The position of the FISH probe used to visualize the IPA RNA is marked in gray and the position of the 3′ cleavage site in the intron is as indicated. ( F ) U2OS cells were transfected with the CCDC71-IPA reporter and, 18 to 24 hours later, the cells were fixed. The IPA transcript was visualized by FISH and nuclear speckles were visualized by immunofluorescence against SC35. Representative images are shown with a merged overlay showing the CCDC71-IPA mRNA in red and SC35 in green. Scale bar, 10 µM. Examples of CCDC71-IPA /SC35 colocalization are indicated with arrows. ( G , H ) Control- and ZFC3H1-depleted cells were transfected with the CCDC71-IPA reporter and the IPA transcript was visualized by FISH. ZFC3H1 depletion increased the cytoplasmic accumulation of the CCDC71-IPA . Representative images are shown in G (scale bar, 10 µm) and quantification is shown in H . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for H. (**) P < 0.01. ( I – K ) ePAT assay and 3′RACE were used to examine 3′ end processing. ( I ) Schematic of the ePAT assay as described in . The ftz -specific ( F′ ) and universal ( R′ ) primers used to amplify the ePAT amplicon are indicated. The sequence of the ePAT amplicon before the cleavage site is shown in J and is 117 nt long. ( J ) The sequence of the end of the 3′UTR is shown. Indicated in bold are the ftz -specific F ′ primer annealing site (used in the ePAT and 3 ′ RACE experiments), the hexanucleotide motif, and the cleavage site (as determined by 3 ′ RACE experiments on mRNAs derived from U2OS cells transfected with either ftz-Δi or ftz-Δi-5′SS ). ( K ) PCR products from the ePAT assay were separated on a 1% agarose gel and stained with ethidium bromide. Lane 1 : Molecular weight markers with sizes in bp indicated on the left ; lanes 3–6 : ePAT amplicons from U2OS cells that were transfected with plasmids containing the indicated versions of the ftz reporter (without [ Δi ] or with [ i ] an intron, without or with the 5′SS motif). Note that the amplicons generated from all four reactions are the same length (∼230 nt). Since the amplified region in the 3 ′ UTR is 117 bp long (see J ), and the universal primer has a 14 nt extension (see J ), the poly(A)-tail is estimated to be ∼100 nt long.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, shRNA, Transduction, SDS Page, Construct, Transfection, Plasmid Preparation, Immunofluorescence, Amplification, Sequencing, Derivative Assay, Agarose Gel Electrophoresis, Staining, Molecular Weight, Generated

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 and U1-70K function in the same pathway for the nuclear retention of 5′SS motif containing mRNAs. ( A ) U2OS cells were treated with lentivirus shRNA against either U1-70K, ZFC3H1, or a mixture of the two. Lysates were collected 96 h post-transduction, separated by SDS-PAGE and immunoprobed for U1-70K, ZFC3H1, and mAb414. Note that to effectively deplete U1-70K, cells were treated with lentivirus containing four shRNA plasmids. Also note that the asterisk (*) denotes a non-specific band. ( B , C ) Control-, U1-70K-, ZFC3H1-, or codepleted cells were transfected with the intronless ftz reporter ± 5′SS as described in . Note that the cytoplasmic/nuclear distribution of ftz-Δi-5′SS mRNA in cells codepleted of U1-70K and ZFC3H1 resembles the distribution in single depletion cells, suggesting that both proteins function in the same pathway. Representative images are shown in C (scale bar, 10 µm) and quantification is shown in D . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for C . (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( D ) HEK cells expressing carboxy-terminally tagged ZFC3H1 (ZFC3H1-FLAG) were lysed and subjected to immunoprecipitation reactions with FLAG M2 beads or mouse IgG (“Control IP”). Immunoprecipitates were separated by SDS-PAGE and immunoprobed for FLAG and U1-70K. For comparison, 1% of the input lysate was also analyzed. The full-length ZFC3H1-FLAG protein is denoted by the asterisk (*) and a shorter band, likely a degradation product, is denoted by the pound (#) sign.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: shRNA, Transduction, SDS Page, Control, Transfection, Expressing, Immunoprecipitation, Comparison

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 and U1-70K are required for nuclear retention of 5′SS motif containing mRNAs in speckles. ( A , B ) Control or ZFC3H1-depleted U2OS cells were microinjected with plasmids containing the βG-Δi reporter ± the 5′SS motif. After the indicated times, the cells were fixed and stained for βG mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. ( A ) Example images of U2OS cells fixed 2 h post-injection with βG reporter mRNA ± the 5′SS motif, with each row representing a single field of view with white arrows pointing to examples of βG mRNA/SC35 colocalization. The merged overlayed image shows βG mRNA in red, SC35 in green. ( B ) Quantification of the degree of βG mRNA/SC35 colocalization in cells depleted of ZFC3H1 or control shRNA treatment by Pearson correlation coefficient analysis as previously described . Each bar represents the average and standard error of three independent experiments, each experiment consisting of 150 to 200 nuclear speckles from 15 to 20 cells. Note that ZFC3H1 is not required for the targeting of 5′SS motif containing mRNAs to nuclear speckles. ( C – E ) Control, U1-70K- or ZFC3H1-depleted U2OS cells were transfected with ftz-Δi ± 5 ′ SS motif. Eighteen to twenty-four hours post-transfection, the cells were fixed and stained for ftz mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. Representative images, with each row depicting a single field of view, is shown in C with merged overlays showing ftz mRNA in red and SC35 in green. Scale bar, 10 µM. Examples of ftz /SC35 colocalization are indicated with arrows. ( D ) The degree of ftz /SC35 colocalization by Pearson correlation coefficient analysis was quantified as above, except that values <0.25 were counted as “not colocalized.” Note that U1-70K or ZFC3H1 depletion leads to decreased level of colocalization between ftz mRNA and SC35. Each bar represents the average and standard error of three independent experiments, each experiment consisting of 100–200 nuclear speckles from 10 to 20 cells. ( E ) The amount of ftz reporter mRNA present in nuclear speckles as a percentage of either the nuclear (“Spec/Nuc”) or total cellular (“Spec/Total”) mRNA levels in transfected cells. Each data point represents the average and standard error of the mean of at least three independent experiments, each experiment consisting of 10–20 cells. Student's t -test was performed, (**) P < 0.01, (***) P < 0.001.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, Staining, Marker, Immunofluorescence, Injection, shRNA, Transfection

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Kinetics of c-Src auto-phosphorylation. (A) Schematic diagram of the functional domains and auto-phosphorylation sites of c-Src. Surface representation of c-Src in closed autoinhibited and open (active) states, current paradigm for c-Src activation and regulation. (B) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–90 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine quantification (total), data represent mean ± SEM of 6 experiments (n=6). (C) Enzymatic assay performed with c-Src WT (3D-construct, 1 μM) incubated with increasing concentrations of ATP at a fixed concentration (1.5 mg/ml) of c-Src Y419 (IEDNEYTARQG) or Y530 (STEPQYQPGEN) derived peptides. Data represent the mean ± SEM, of 2 experiments (n=2) in duplicate. Enzymatic activity (ODs -1 x 10 −3 ). Catalytic efficiency constants (k cat /K M , fold difference) are depicted in the panel below. (D) Mass spectrum of the [M + 2H] +2 ion (m/z 772.3) of a peptide phosphorylated on Tyr 530 (90 min). Below, phosphorylation kinetics of Tyr419 and Tyr530 phospho-peptides measured by mass spectrometry (0-90 min) are depicted. Data represent the mean of normalized phospho-signal ± SEM of 2 experiments (n=2), with two technical replicates each.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Functional Assay, Activation Assay, Construct, Staining, Enzymatic Assay, Incubation, Concentration Assay, Derivative Assay, Activity Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Activation-loop Tyr 419 controls c-Src susbtrate specificity. (A) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT, Y419F and Y530F (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-signal quantification (total phospho-tyrosine), data represent mean ± SEM of 6 experiments (n=6) in duplicate. (B) Enzymatic assay performed with c-Src WT, Y419F and Y530F (3D-construct, 1 μM) incubated with increasing concentrations of ATP at a fixed concentration (4 mg/ml) of peptide: ABL derived-peptide (EAIYAAPFAKKK) or RET activation loop Y905 derived-peptide (DVYEEDSFVK). Data represent the mean ± SEM, of 4-6 experiments (n =4-6) in duplicate. Catalytic efficiency constants (k cat /K M , fold difference) are depicted in the right panel. (C-F) WB of samples from a time course phosphorylation assays with c-Src (3D, 1 μM) WT, Y419F and Y530F and using the following substrates surrogates: FAK (aa 1-405), c-Src KD K298M, RET KD (aa 713-1012) K758M and deltaKIF5B-RET, respectively using the indicated antibodies. (G) Logo consensus sequence for optimal c-Src susbtrate, from PhosphoSitePlus (CST).

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Activation Assay, Construct, Staining, Enzymatic Assay, Incubation, Concentration Assay, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Dissecting cis-versus-trans components for c-Src auto-phosphorylation. (A) WB of samples from a time-course auto-phosphorylation experiment with WT, Y419F and Y530F c-Src (3D-construct, 0.25-2.5 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated c-Src phospho-Tyr 419 antibody. Total amount of protein was visualized by Coomassie staining. Diagram for the in-trans and cis-mechanism for auto-phosphorylation, lower inset. (B) WB of samples from a time-course phosphorylation assay (0-90 min) as in A, in the absence and in the presence of a c-Src KD K298M construct as an intact susbtrate surrogate using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Diagram for the enzyme (E) and susbtrate (S) acting kinases. lower inset. (C) WB of samples from a time-course experiment with c-Src KD WT in the presence of c-Src 3D-K298M constructs with and without Y419F and Y530F mutations as substrates using the indicated antibodies. Total c-Src protein was visualized by Coomassie staining. Diagram for the enzyme (E) and susbtrate (S) acting kinases. lower inset.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Construct, Staining, Phosphorylation Assay

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: (A) Schematic diagram of the functional domains and main auto-phosphorylation sites of c-Src. Different c-terminal sequence variants are depicted. (B) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT, v-Src and 531X (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine 216 quantification signal, data represent mean ± SEM of 3 experiments (n=3), * p < 0.05, one-way ANOVA test. (C) Enzyme kinetics and catalytic efficiency constant (k cat /K M , fold-change) for ATP using Src WT, v-Src and 531X (3D-construct, 1 μM final concentration) at fixed concentration (2-4 mg/ml) of Abl peptide. Data represent the mean ± SEM, of 3 experiments in duplicate (n=3). (D) Cartoon representation of the kinase-susbtrate (chain A and B) engagement of two c-Src molecules in the crystal structure. Left panel, close in view of the c-terminal aa sequence of c-Src containing the palindromic PQYQP motif at the interface between the two molecules showing residues coordinated by intra- and inter-molecular interactions. (E) WB of samples from a time-course phosphorylation experiment with c-Src WT, v-Src and 531X (3D-construct, 1 μM) in the presence of a c-Src KD K298M (3 μM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. (F) WB of samples from a time-course phosphorylation experiment with c-Src KD WT (1 μM), in the presence of susbtrate surrogates c-Src, v-Src and 531X K298M (3D-construct, 3 μM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine 419 and 530 quantification signal on the active kinase molecule, data represent mean ± SEM of 3 experiments (n=3), *** p = 0.0005, **** p = 0.0001, 2-way ANOVA test.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Functional Assay, Sequencing, Construct, Staining, Concentration Assay