Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 2. Liprin-a1, ERC1a and LL5 proteins regulate the stability of lamellipodia. (A) Time lapse of MDA- 231 cells on fibronectin. White arrows indicate the appearance of new protrusions in cells transfected with the indicated siRNAs. Scale bar: 10 mm. (B) Liprin-a1, ERC1a or LL5 silencing (Lip, liprin-a1; ERC, ERC1a; LL5s, both LL5a and LL5b) in cells migrating on fibronectin. Lamellipodial stability is reduced compared to transfection with control siRNA (Luc). Results are mean6s.e.m. (n510 cells). (C) Cells overexpressing liprin-a1 (L4) produce less frequent and more stable lamellipodia compared to control (C9) cells. Effects of liprin-a1 overexpression are abolished by silencing liprin-a1, ERC1a or LL5 proteins. Results are mean6s.e.m. (n510 cells). (D) Overexpression of ERC1a or LL5b decreases the frequency of lamellipodia formation and increases lamellipodial persistence (n55–10 cells). (E) ERC1a overexpression rescues the frequency of lamellipodia formation (top) and partially rescues the loss of their persistence (bottom) induced by liprin- a1 silencing. Results are mean6s.e.m. (n59–17 cells). *P,0.05; **P,0.005 compared with control or as indicated.

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Transfection, Control, Over Expression

Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 3. ERC1a colocalizes with liprin-a1 and LL5b at the edge of migrating cells. MDA-231 cells migrating on fibronectin were immunostained for the endogenous proteins: colocalization at protrusions (arrows) of ERC1a, liprin-a1 and LL5a (A); ERC1a with LL5b and LL5a (B); ERC1a, liprin-a1 and LL5b (C). Scale bars: 10 mm. Lower panels in A and C are threefold enlargements of areas indicated by arrowheads. Arrows indicate examples of colocalization. (D) Frames from cells expressing the indicated proteins and migrating on fibronectin. GFP-tagged liprin-a1, ERC1a, LL5a and LL5b are concentrated in clusters near the leading edge of migrating cells. Cells cotransfected with GFP and Mito-RFP (Mitochondria-RFP) were used as controls. Scale bar: 10 mm. (E) Frames from a time- lapse series of a cell co-transfected with mCherry and GFP–ERC1a. Scale bar: 20 mm. (F) Kymograph for mCherry and GFP–ERC1a from the lamellipodium of the cell shown in E (white line) showing intense accumulation of ERC1a near the lamellipodium during the protrusive phase. The signal for ERC1a decreases abruptly once the lamellipodium halts (red arrow) and retracts (blue arrow). Right, diagram of the intensity of fluorescence for ERC1a (green line) and mCherry (red line). The intensity of ERC1a signal increases soon after lamellipodial protrusion is detectable (red line) and abruptly decreases when the protrusive activity stops.

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Expressing, Transfection, Fluorescence, Activity Assay

Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 5. Silencing of liprin-a1, ERC1a or LL5 proteins enhances the intracellular accumulation of total b1 integrins. (A) ERC1a-positive (ERC) clusters at protrusions are contiguous, but do not overlap, with paxillin-positive focal adhesions. Scale bars: 10 mm. (B) Frames from time-lapse of a cell co-transfected with mCherry–zyxin and GFP–ERC1a. ERC1a accumulates behind peripheral focal adhesions at the front of the migrating cell. Scale bar: 10 mm. (C,D) Cells co- transfected with GFP and the indicated siRNAs (Lip, siLip, liprin-a1; siERC1a, ERC1a; LL5s, siLL5a+b, both LL5a and LL5b) were incubated with the mAb TS2/16 before fixation for immunofluorescence. Merge: GFP (green), integrins (red), DAPI (blue). Results are mean6s.e.m. for the normalized density of b1 integrin at the cell surface (n536–47 cells). (E,F) Cells transfected with GFP and siRNAs were incubated overnight at 37˚C with the TS2/16 mAb. Surface labeling of integrins was removed before fixation and detection of the internalized integrins with fluorescently tagged anti-mouse-IgG antibody. Scale bar: 20 mm (C,E). Silencing of any of the three proteins enhanced total b1 internalization (F). Bars, normalized mean6s.e.m. (n563–85 cells); *P,0.05; **P,0.005 compared with control.

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Transfection, Incubation, Immunofluorescence, Labeling, Control

Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 8. The PH domain of LL5b is required for effective migration, and for the localization of endogenous liprin-a1 and ERC1a near the protruding cell edge. (A) siRNAs specific for endogenous LL5 proteins (LL5s) (upper two blots from top) do not interfere with the expression of GFP-tagged mouse wild- type and mutant LL5b (lowest blot). Immunoblotting was performed on lysates (15 mg protein) from transfected cells to detect the proteins indicated on the left of each blot. The anti-LL5b antibody recognizes the endogenous human protein, but not the murine overexpressed protein. Luc, luciferase. (B,C) Subcellular localization of endogenous liprin-a1 in cells co-transfected with siRNAs for either control or LL5 proteins, together with the indicated GFP constructs. Scale bars: 20 mm. (D) Effects of the expression of wild-type LL5b or the mutant LL5bM on the accumulation of endogenous liprin-a1 at the cell edge. Results are mean6s.e.m. (n545–76 cells). *P,0.05; **P,0.005 compared with control. (E,F) Localization of endogenous ERC1a in cells co-transfected with siRNAs for either control or LL5 proteins, together with the indicated GFP constructs. Scale bars: 20 mm. (G) The negative effects of LL5 proteins depletion on the speed of migration on fibronectin are rescued by GFP–LL5b, but not by GFP–LL5bM. Results are mean6s.e.m. (n542–81 cells). The effects of LL5 proteins depletion on the formation (H) and the persistence (I) of lamellipodia during migration on fibronectin are rescued by the wild-type GFP–LL5b. The GFP–LL5bM

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Migration, Expressing, Mutagenesis, Western Blot, Transfection, Luciferase, Control, Construct

Journal: Nature communications

Article Title: PPFIA1 drives active α5β1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis.

doi: 10.1038/ncomms13546

Figure Lengend Snippet: Figure 6 | Fibrillar adhesions and FN fibrils are dynamic structures governed by Rab21 and PPFIA1. (a) Living siCTL and siRAB21 ECs were incubated with IST9 mAb for 30 min, fixed, acid washed and stained. RAB21 silencing impairs ED-A FN endocytosis as revealed by the decrease of IST9 punctae co-localizing with the endosome marker EEA1. Right panels are magnifications of the boxed areas in left panels. Data are mean value±s.e.m., n ¼ 70 cells per condition pooled from two independent experiments. ***Po0.001. (b) Left panel, western blot analysis of PPFIA1, RAB21 and actin on total lysates of siCTL, siPPFIA1 and siPPFIA1 þ siRAB21 ECs. Right panel, western blot analysis of the insoluble matrix fraction of ECs that were extracted with DOC buffer. PPFIA1 silencing dramatically reduces the amount of DOC-insoluble fraction of endogenous ED-A FN. Of note, simultaneous silencing of Rab21 GTPase (siPPFIA1 þ siRAB21), which drives integrin endocytosis, rescues the defective incorporation of endogenous ED-A FN in the DOC-insoluble fraction of siPPFIA1 ECs. (c) Confocal microscopy analysis of the patterning of endogenous cellular ED-A FN (green) in fixed confluent ECs. Before fixation, living ECs were incubated for 20 min with exogenous SNAKA51 (red). ED-A FN polymerizes into a fibrillar network in siCTL, but not in siPPFIA1 ECs. Simultaneous silencing of Rab21 GTPase (siPPFIA1 þ siRAB21) fully restores ED-A FN polymerization in siPPFIA1 ECs. SNAKA51 þ active a5b1 integrin localizes in fibrillar adhesion in siCTL, but not in siPPFIA1 ECs. Notably, simultaneous Rab21 (siPPFIA1 þ siRAB21) silencing promotes the localization of SNAKA51 in fibrillar adhesion of siPPFIA1 ECs. Data are mean±s.e.m., n ¼ 20 cells per condition pooled from two independent experiments. Scale bar, 20 mm (a,c, right), 50mm (c, left). ***Po0.001; Student’s t-test.

Article Snippet: Human PPFIA1 cDNA (transcript variant 1, NM_003626) from Origene Technologies Rockville, MD, USA was used as a template.

Techniques: Incubation, Staining, Marker, Western Blot, Confocal Microscopy

Journal: Frontiers in immunology

Article Title: Identification of a Novel Serological Marker in Seronegative Rheumatoid Arthritis Using the Peptide Library Approach.

doi: 10.3389/fimmu.2021.753400

Figure Lengend Snippet: FIGURE 4 | Peptides cross-reaction of RA patients’ sera and recognition of auto-antigens by purified anti-RA-peptide Abs. (A) Frequency of the direct binding of seronegative and seropositive patients’ sera to RA, Pyk/Fadk2, Bank-1, Liprin-1 and CTLA-4 peptides evaluated by ELISA assays. No binding was observed using serum IgG of healthy donors. The arbitrary cutoff of 0.090 was used. (B) Immunoblot analysis of cell lysates, after immunoprecipitation with affinity purified anti-RA- peptide antibodies detected with commercial antibodies: mouse polyclonal antibodies directed against PYK2 or rabbit polyclonal antibodies directed against BANK, or rabbit polyclonal antibodies directed against Liprin. PBMCs, T or B cells were obtained by a normal healthy donor; HL60 (Human promyelocytic leukemia) and DAUDI (Burkitt lymphoma cell) lines were used as controls; HLFS (Normal Human Fibroblast-Like Synoviocytes) and HLFS-RA (Human Fibroblast-Like Synoviocytes: Rheumatoid Arthritis) and NHDF (Normal human dermal fibroblast) were commercially obtained.

Article Snippet: Immunoblots were performed using the following primary antibodies: anti-RA specific peptide antibodies, mouse polyclonal antibodies directed against PYK2 (Novus Biologicals, Littleton, CO, USA), rabbit polyclonal antibodies directed against Liprin (Novus Biologicals Littleton, CO, USA) and rabbit polyclonal antibodies directed against Bank (Abgent, San Diego, CA, USA) followed by their specific peroxidaselinked secondary antibodies.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation