Journal: Frontiers in Immunology

Article Title: Feeder-cell-free system for ex vivo production of natural killer cells from cord blood hematopoietic stem and progenitor cells

doi: 10.3389/fimmu.2025.1531736

Figure Lengend Snippet: Antibody panels used for flow cytometry.

Article Snippet: hPerforin , PE , REA1061 , Miltenyi , 130-118-117.

Techniques: Cytometry

Journal: PLoS ONE

Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137

doi: 10.1371/journal.pone.0032802

Figure Lengend Snippet: A , Equal amounts of HEK293 cell lysate were separated on SDS-PAGE, and individual lanes were blotted with P3490 that was pre-incubated with no peptide (Lane 1), or PFN1 peptides containing unphosphorylated (Lane 2) or phosphorylated Ser-137 (Lane 3). PFN1-pS137 peptide completely blocked the binding of P3490 to PFN1. Unphosphorylated PFN1-S137 peptide blocked ∼90% binding vs. the no peptide control. Total PFN1 (on a separate and identically loaded gel) and actin levels were confirmed as being equal across all lanes. B , P3490 stains the cytoplasm of multiple cell lines in a heterogeneous fashion, including HEK293, HeLa and NIH 3T3 (in green). DAPI was used as counterstain (in blue). C , Immunostaining of HEK293 cells with P3490 (green) was unaffected by pre-incubation with the unphosphorylated PFN1-S137 peptide, but was completely inhibited by the phosphorylated PFN1-pS137 peptide. DAPI staining is shown in blue. D , NIH 3T3 cells were serum-starved for 16 hr in the absence or presence of 50 µM Y-27632, followed by treatment with 10 µM Lysophosphatidic acid (LPA) for 60 min. Cells were immunostained with P3490 (green) and counterstained with DAPI (blue) (a–c). LPA increased P3490 staining, which was blocked by the pretreatment with Y-27632. Total PFN1 levels were unaffected by these treatments as indicated by immunostaining with a generic PFN1 antibody (PFN1 in green and DAPI in blue) (d–l). E , HEK293 cells were treated with or without 50 µM hydroxyfasudil (OH-fasudil) for 2 hr, followed by immunostaining with P3490 (a–b) or the generic PFN1 antibody (c–d) (both in green) and counterstaining with DAPI (blue). Nearly all cells treated with OH-fasudil became P3490-negative without changes in their total PFN1 levels. F , HEK293 cells grown in a 96-well plate were treated for 24 hr with increasing concentrations of OH-fasudil (1, 3, 10, 50 µM), immunostained with P3490 and Alexa Fluor®488-labeled secondary antibody, and counterstained with DAPI. Fluorescence intensity was quantified on a fluorescence plate reader. P3490 staining was normalized vs. DAPI to control for cell numbers, and the effects of OH-fasudil were calculated as relative values as compared to the untreated cells. OH-fasudil reduced P3490 staining dose-dependently. Error bars represent the standard error of the mean (SEM). Data are mean ± SEM of three independent experiments.

Article Snippet: Custom-made antibodies include rabbit polyclonal antibody against the C-terminus of PFN1 (KCYEMASHLRRSQY, gift of Dr. Nicholas A. DiProspero) and affinity purified rabbit polyclonal anti-pS137-PFN1 (New England Peptides, Ac-CMASHLRR(pS)QY-OH) .

Techniques: SDS Page, Incubation, Binding Assay, Control, Immunostaining, Staining, Labeling, Fluorescence

Journal: PLoS ONE

Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137

doi: 10.1371/journal.pone.0032802

Figure Lengend Snippet: A , HEK293 cells were infected for three days with lentiviral shRNA particles encoding either a scrambled nucleotide sequence (Ctrl shRNA), or a target-specific sequence for human PFN1 (PFN1 shRNA). Efficient PFN1 knockdown was confirmed by Western blot. B , virus-mediated PFN1 knockdown reduced P3490 staining (green, a–b), as visualized by confocal fluorescence microscope. Total PFN1 decrease was also confirmed by immunofluorescence staining (green, c–d). DAPI (blue) was used to counterstain cells either as merged (a–b) or unmerged images (e–f). C , staining of virus-infected cells with P3490 and PFN1 antibodies (as in A and B) was quantified by fluorescence plate reader, and normalized vs. DAPI to control for cell numbers. The decrease of P3490 (∼40%; *, p<0.05, unpaired t-test) and total PFN1(60%; p<0.001, unpaired t-test) staining caused by PFN1 knockdown was calculated relative to cells infected with the control shRNA (arbitrarily set as 1). Error bars represent the standard error of the mean (SEM). Data are mean ± SEM of three independent experiments. D , HEK293 cells were transiently transfected with Myc-tagged PFN1(wt, S137A or S137D), treated with 50 µM hydroxyfasudil for 16 hr, and double stained with an anti-Myc (red) and P3490 (green) antibodies. Only cells expressing the phosphomimetic Myc-PFN1(S137D) stained positive with P3490 (g–i), but not Myc-PFN1(wt) (a–c) or Myc-PFN1(S137A) (d–f). Arrowheads indicate two cells that expressed Myc-PFN1(S137D) and stained positive with P3490. E , Western blot confirmed comparable levels of Myc-PFN1 over-expression (wt, S137A and S137D) in transiently transfected HEK293 cells, as compared to cells transfected with an empty vector (pcDNA3). F , cells transfected (as in E) with pcDNA3 or various PFN1 constructs were immunostained with P3490, and quantified on a fluorescence plate reader with normalization vs. DAPI. The effects of PFN1 over-expression on P3490 staining are represented as relative change compared to cells transfected with pcDNA3. Only PFN1(S137D) increased P3490 staining (***, p<0.001, unpaired t-test). Cells were not treated with hydroxyfasudil. Error bars represent the SEM. Data are mean ± SEM of three independent experiments.

Article Snippet: Custom-made antibodies include rabbit polyclonal antibody against the C-terminus of PFN1 (KCYEMASHLRRSQY, gift of Dr. Nicholas A. DiProspero) and affinity purified rabbit polyclonal anti-pS137-PFN1 (New England Peptides, Ac-CMASHLRR(pS)QY-OH) .

Techniques: Infection, shRNA, Sequencing, Knockdown, Western Blot, Virus, Staining, Fluorescence, Microscopy, Immunofluorescence, Control, Transfection, Expressing, Over Expression, Plasmid Preparation, Construct

Journal: PLoS ONE

Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137

doi: 10.1371/journal.pone.0032802

Figure Lengend Snippet: A , HEK293 cells were treated with okadaic acid (OA) (a–d) or endothall (e–h) for 16 hr at increasing concentrations (1, 3 and 10 nM for OA; 1, 3 and 10 µM for endothall), followed by immunostaining with P3490 (green) and counterstaining with DAPI (blue). The number of P3490-positive cells and overall P3490 staining intensity increased notably at 10 nM of OA and 10 µM of endothall treatment. B , effects of OA and endothall on P3490 staining were quantified using a fluorescence plate reader. P3490 levels were normalized vs. DAPI, and the effects of the drugs were represented as relative change vs. the vehicle controls. OA and endothall increased P3490 staining dose-dependently. Error bars represent the SEM. Data are mean ± SEM of three independent experiments. C , HEK293 cells treated with 10 nM OA (a–b) or 10 µM endothall (c–d) were immunostained for total PFN1 (green) and counterstained with DAPI (blue). Merged images showed no changes in total PFN1 levels as a result of the drugs.

Article Snippet: Custom-made antibodies include rabbit polyclonal antibody against the C-terminus of PFN1 (KCYEMASHLRRSQY, gift of Dr. Nicholas A. DiProspero) and affinity purified rabbit polyclonal anti-pS137-PFN1 (New England Peptides, Ac-CMASHLRR(pS)QY-OH) .

Techniques: Immunostaining, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137

doi: 10.1371/journal.pone.0032802

Figure Lengend Snippet: A , HEK293 cells were transfected with control, or sequence-specific siRNAs targeting human PP1Cα or PP2ACα. They were immunostained with P3490 (a–c) or the antibody against total PFN1 (d–f) (both in green) and counterstained with DAPI (blue). Silencing PP1Cα, but not PP2ACα, increased the number of P3490-positive cells and their staining intensities, while having no effect on total PFN1 levels. B , effects of PP1Cα and PP2ACα knockdown on P3490 staining were quantified using the fluorescence plate reader, normalized vs. DAPI, and represented as change relative to control siRNA. PP1Cα knockdown increased P3490 staining three-fold (***, p<0.001, unpaired t-test), while PP2ACα knockdown had no effect. Error bars represent the SEM. Data are mean ± S.E.M. of three independent experiments. C , Western blot confirmed >90% knockdown of both PP1Cα and PP2ACα in HEK293 cells. Neither affected PFN1 levels. Arrowhead indicates PP1Cα. The identity of the protein band above PP1Cα, which cross-reacts with PP1Cα antibody, is unknown. D , HeLa cells were transfected with Myc-PP1Cα(wt) (a–c), Myc-PP1Cα(H125A) (catalytically inactive) (d–f) or Myc-PP2ACα (g–i), double immunolabeled with an anti-Myc antibody (red) and P3490 (green), and counterstained with DAPI (blue). Neither Myc-PP1Cα(H125A) nor Myc-PP2ACα over-expression affected P3490 staining of the transfected cells (as indicated by the presence of cells that were both red and green). However, PP1Cα(wt) over-expression inhibited P3490 staining (as indicated by the mutual exclusivity of red and green cells).

Article Snippet: Custom-made antibodies include rabbit polyclonal antibody against the C-terminus of PFN1 (KCYEMASHLRRSQY, gift of Dr. Nicholas A. DiProspero) and affinity purified rabbit polyclonal anti-pS137-PFN1 (New England Peptides, Ac-CMASHLRR(pS)QY-OH) .

Techniques: Transfection, Control, Sequencing, Staining, Knockdown, Fluorescence, Western Blot, Immunolabeling, Over Expression

Journal: PLoS ONE

Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137

doi: 10.1371/journal.pone.0032802

Figure Lengend Snippet: A , HEK293 cells were co-transfected with an untagged PFN1 along with either an empty vector (pcDNA3) (Lane 1), or Myc-tagged PP1Cα (Lane 2), or PP2ACα (Lane 3), followed by immunoprecipitation with an anti-Myc antibody and Western blot for PFN1. PFN1 co-immunoprecipitated specifically with PP1Cα, but not PP2ACα. B , HA-PFN1 constructs (wt, S137A or S137D) were co-transfected into HEK293 cells with pcDNA3 (Lanes 1–3) or Myc-PP1Cα (Lanes 4–6). Myc-PP1Cα was immunoprecipitated with an anti-Myc antibody, and co-immunoprecipitated HA-PFN1 was detected by an anti-HA antibody. Relative to PFN1(wt), more PFN1(S137D) and less PFN1(S137A) bound to PP1Cα. C , Purified 6His-PFN1 (wt, S137A or S137D) proteins were bound to Ni-NTA beads, and subsequently mixed with recombinant PP1Cα (Lanes 2–4). Beads without His-PFN1 were mixed with the same amount of PP1Cα to control for nonspecific binding (Lane 1). Beads were washed, and analyzed for binding of PP1Cα to 6His-PFN1 by Western blot. 6His-PFN1 proteins were visualized by Coomassie blue staining. No specific binding was observed between purified PP1Cα and 6His-PFN1 (wt, S137A or S137D), despite that they were abundantly present.

Article Snippet: Custom-made antibodies include rabbit polyclonal antibody against the C-terminus of PFN1 (KCYEMASHLRRSQY, gift of Dr. Nicholas A. DiProspero) and affinity purified rabbit polyclonal anti-pS137-PFN1 (New England Peptides, Ac-CMASHLRR(pS)QY-OH) .

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Construct, Purification, Recombinant, Control, Binding Assay, Staining