Journal: Life Science Alliance

Article Title: New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

doi: 10.26508/lsa.202101301

Figure Lengend Snippet: (A) Alignment of cytoplasmic amino acid residues of mouse integrin β subunits. The conserved threonine motif (red), the proximal NPxY motif (green), the distal NxxY motif (yellow-green), and the binding sites of talin, kindlin-2, SNX17, and filamin-A are indicated. (B) Sequences of the synthetic peptides used to determine the specificity of the anti-β1-pTpT antibodies by ELISA. (C) Affinity measurement of the anti-β1-pTpT integrin antibody (Thermo Fisher Scientific) to the immobilized β1-pTpT, β1-TT/AA, β1-TT/DD, and β1-WT tail peptides. The dilution factor of the antibody used to generate the titration curve is indicated on the plot. The EC 50 dilution is indicated. (D, E) Quantification of binding of the anti–β1- p T p T integrin antibody (Thermo Fisher Scientific) (D) and anti–β1- p T p T integrin antibody (Abcam) (E) towards the indicated β1 integrin tail and phosphorylated Wwc2 peptides. A dilution range between 1:500 and 1:10,000 of the antibodies was used.

Article Snippet: The following antibodies or molecular probes were used at indicated concentrations for Western blot (W), immunofluorescence (IF), or FC: kindlin-2 (MAB2617 from Millipore) W: 1:1,000; talin (8D4 from Sigma-Aldrich) W: 1:1,000; paxillin (610051, clone 349 from BD Transduction Laboratories) IF: 1:500; paxillin (sc-5574 from Santa Cruz Biotechnology) IF: 1:200; integrin β1-Alexa 488-conjugated (102211 from BioLegend) FC: 1:300; integrin β1 (home-made [ ]) W: 1:10,000, IF: 1:4,000, IP: 2 μl/mg lysate; integrin β1 (clone MB1.2 from Millipore) IF: 1:500; integrin β1 (clone JB1B from Novus Biologicals) IP: 5 μl/mg lysate; integrin α5-PE (557446 from Pharmingen) FC: 1:300; IgG2a rat isotype control (13-4321 from eBioscience) FC: 1:300; pT788/789 β1-integrin (44-872G from Thermo Fisher Scientific) W: 1:1,000, IF 1:300; pT788/789 β1-integrin (ab5189 from Abcam) W: 1:1,000, IF 1:300, phospho-threonine (clone 20H6.1 from Merck) W: 1:1,000, IF 1:500, IP: 5 μl/mg lysate; phospho-threonine (clone RM102 from Thermo Fisher Scientific) W: 1:1,000, IF 1:300, IP: 5 μl/mg lysate; PPM1F ( ) W: 1:300; SNX17 (10275-1-AP from Proteintech) W: 1:1,500.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Titration

Journal: Life Science Alliance

Article Title: New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

doi: 10.26508/lsa.202101301

Figure Lengend Snippet: (A) Streptavidin-bead pull-down assay with the indicated biotinylated β1-integrin cytoplasmic tail (β1-tail) peptides and recombinant kindlin-2, or a combination of kindlin-2 and talin head domain. After pull-down, bound proteins were detected by Western blotting. (B) Densitometric analysis of kindlin-2 binding to indicated β1-tail peptides in the absence and presence of talin head domain. Data are mean ± SD. n = 3. ns, not significant (Mann–Whitney U test). (C) Affinities of talin and kindlin-2 for indicated Atto488-labeled β1-tail peptides determined by microscale thermophoresis. (D) Indicated biotinylated β1-tail peptides were analyzed for binding to endogenous talin, kindlin-2, and SNX17 by Western blotting. Wcl, whole cell lysate. (E) Densitometric analysis of talin, kindlin-2, and SNX17 to the indicated β1-tail peptides. Data are mean ± SD of n = 5 independent experiments. ** P < 0.01; ns, not significant (Mann–Whitney U test). Statistical significances are calculated between β1-WT and mutant β1-tail peptides.

Article Snippet: The following antibodies or molecular probes were used at indicated concentrations for Western blot (W), immunofluorescence (IF), or FC: kindlin-2 (MAB2617 from Millipore) W: 1:1,000; talin (8D4 from Sigma-Aldrich) W: 1:1,000; paxillin (610051, clone 349 from BD Transduction Laboratories) IF: 1:500; paxillin (sc-5574 from Santa Cruz Biotechnology) IF: 1:200; integrin β1-Alexa 488-conjugated (102211 from BioLegend) FC: 1:300; integrin β1 (home-made [ ]) W: 1:10,000, IF: 1:4,000, IP: 2 μl/mg lysate; integrin β1 (clone MB1.2 from Millipore) IF: 1:500; integrin β1 (clone JB1B from Novus Biologicals) IP: 5 μl/mg lysate; integrin α5-PE (557446 from Pharmingen) FC: 1:300; IgG2a rat isotype control (13-4321 from eBioscience) FC: 1:300; pT788/789 β1-integrin (44-872G from Thermo Fisher Scientific) W: 1:1,000, IF 1:300; pT788/789 β1-integrin (ab5189 from Abcam) W: 1:1,000, IF 1:300, phospho-threonine (clone 20H6.1 from Merck) W: 1:1,000, IF 1:500, IP: 5 μl/mg lysate; phospho-threonine (clone RM102 from Thermo Fisher Scientific) W: 1:1,000, IF 1:300, IP: 5 μl/mg lysate; PPM1F ( ) W: 1:300; SNX17 (10275-1-AP from Proteintech) W: 1:1,500.

Techniques: Pull Down Assay, Recombinant, Western Blot, Binding Assay, MANN-WHITNEY, Labeling, Microscale Thermophoresis, Mutagenesis

Journal: bioRxiv

Article Title: Phostensin Enables Lymphocyte Integrin Activation and Population of Peripheral Lymphoid Organs

doi: 10.1101/2021.09.24.461584

Figure Lengend Snippet: (A-B) U2-OS cells that stably express integrin α IIb -SBP(streptavidin-binding peptide) and β 3 (D119A) to form ligand binding-defective α IIb (SBP)β 3 (D119A) were transduced with a lentivirus encoding a shRNA against PTSN prior to transfection with mCherry-tagged β-PTSN in combination with Flag-RIAM. Flag-tagged RIAM was captured by (A) anti-Flag immunoprecipitation and (B) associated proteins were analyzed by immunoblotting. (C-E) Integrated density values for immunoreactive bands corresponding to integrin β 3 (C) , RIAM (D) or talin (E) . Bar graphs represent mean ± SEM (n = 3 independent experiments) normalized to control condition (sh-scrambled). Two-tailed t -test; ns not significant, * p<0.05.

Article Snippet: Antibodies against talin (8d4, Sigma-Aldrich) and Flag (M2) were from Sigma-Aldrich.

Techniques: Stable Transfection, Binding Assay, Ligand Binding Assay, Transduction, shRNA, Transfection, Immunoprecipitation, Western Blot, Two Tailed Test