Journal: Acta neuropathologica communications

Article Title: Trichuris suis induces human non-classical patrolling monocytes via the mannose receptor and PKC: implications for multiple sclerosis.

doi: 10.1186/s40478-015-0223-1

Figure Lengend Snippet: Fig. 6 PKC signals downstream upon TsSP-MR interaction. Quantification of flow cytometry analysis of the expression levels of (a) CCR2, (b) LFA-1 avidity and (c) LFA-1 affinity on human monocytes. These cells were cultured in the presence or absence of TsSP (40 μg/ml, 16 h), the PKC inhibitor Bisindolylmaleimide I (GF109203, 2 μM) and blocking antibodies for the MR or its isotype control (both 10 μg/ml). (b) and (c) are expressed as the percentage of avidity/affinity compared to total LFA-1. Cell supernatants were used to determine the levels of (d) TNF-α and (e) IL-10 secretion by enzyme-linked immunosorbent assay analysis. f PKC activation was determined by Western Blotting using phosphoPKC antibodies compared to GAPDH expression and images were subsequently quantified using ImageJ. Experiments were performed in triplicate using cells derived from 4 different human donors and the results are presented as the mean (a-c); the mean percentage (d, e) or the mean PKC phosphorylation +/−SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by ANOVA and Students t test

Article Snippet: The following primary antibodies (in TSM/0,05 % Tween/5 % BSA) were used: PhosphoPKC (Cell signaling, Beverly, MA, USA, #9371S) and GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-32233) as a loading control.

Techniques: Flow Cytometry, Expressing, Cell Culture, Blocking Assay, Control, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Derivative Assay, Phospho-proteomics

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro.

doi: 10.1007/s00109-022-02280-6

Figure Lengend Snippet: Fig. 1 Expression of Pkm isoforms during cutaneous wound healing. A Quantification of Pkm1 and 2 mRNA expression in murine wounds as assessed by RT-PCR (left panel) and Pkm2 mRNA expression in wound edge (wound margin) and wound bed (inner wound granula- tion tissue) compartments (right panel) at the indicated time points after injury. Non-wounded skin served as a control (ctrl skin). Bars indicate means ± SD obtained from three wounds (n = 3) isolated from four individual animals (n = 4). **P < 0.01, *P < 0.05 (ANOVA) as compared to control skin (Dunnett’s post-hoc test). B Immunob- lot analysis for Pkm isoforms and phosphorylated Pkm(Y105) protein expression in murine wounds (wd) at the indicated time points after

Article Snippet: Mouse monoclonal antibody (mAb) anti-ß-actin (ACTB) (AC-15) (A5441; Sigma), rabbit anti-Akt (9272; Cell Signaling Technology (CST), Frankfurt am Main, Germany), rabbit mAb anti phospho-Akt (Ser473) (D9E) XP® (4060; CST), rabbit mAb anti-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) (53H11) (9644; CST), rabbit anti-phospho-4E-BP1 (Ser65) (9451; CST), rabbit antieukaryotic initiation factor 2 (eIF2) α (9722; CST), rabbit mAb anti-phosphor-eIF2α (Ser51) (119A11) (3597; CST), rabbit anti-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9102; CST), rabbit mAb anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) (4376; CST), rat mAb anti-mouse F4/80 (MCA497; BioRad, Munich, Germany), mouse mAb anti-human GAPDH (GT239) (GTX627408; GeneTex, Biozol, Eching, Germany), mouse mAb anti-human glutamine synthase (6/GS) (610517; Becton Dickinson, Heidelberg, Germany), rabbit anti-mouse Hif-1α (NB100-479; Novusbio Biologicals, Wiesbaden, Germany), rabbit anti-human HIF-1α (10006421; Cayman Chemical Biomol, Hamburg, Germany), goat anti-human lamin B (sc-6216; Santa Cruz Biotechnology, Heidelberg, Germany), rat mAb anti-mouse Ly-6B.2 (7/4) (MCA771; BioRad), rat mAb anti mouse Ly-6G (1A8) (BE0075-1; Bio X Cell, Lebanon, NH, USA), rabbit mAb anti PKM1 (D30G6) XP® (7067; CST), rabbit mAb antiPKM2 (D78A4) XP® (4053; CST), mouse mAb anti-PKM2 (1C11C7) (CoraLite (CL) 488–60268; Proteintech®, Chicago, USA), rabbit anti-phosphoPKM (Y105) (3827; CST), rabbit mAb anti-S6 ribosomal protein (5G10) (2217; CST), rabbit mAb anti-phospho-S6 ribosomal protein (Ser235/236) (2F9) (4856; CST), mouse mAb anti-Stat3 (124H6) (9139; CST), rabbit mAb anti-phospho-Stat3 (Tyr705) (D3A7) (9145; CST), goat anti-VEGF (sc-1836; Santa Cruz).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Impaired T cell protein kinase C delta activation decreases ERK pathway signaling in idiopathic and hydralazine-induced lupus.

doi: 10.4049/jimmunol.179.8.5553

Figure Lengend Snippet: FIGURE 1. Decreased PMA-Raf-1-Erk pathway phosphorylation in lupus T cells. A, CD4 T cells from a healthy control (N) or from two different patients with SLE (L1, L2) were isolated and immediately stimulated with 50 ng/ml PMA for 15 min or not stimulated as described in Materials and Methods. Proteins from whole cell lysates were fractionated by SDS-PAGE, transferred to nitrocellulose membranes and probed with a polyclonal Ab against the active dually phosphorylated form of ERK1/2. The membrane was stripped and reblotted sequentially for phospho-MEK1/2 Ser217/221 and Raf-1 p-Ser338. -actin was used as loading control. B, Quantitative immunoblot analysis of phospho-ERK, phospho-MEK, and phospho-Raf in CD4 T cells from three to six different patients with active lupus treated as in A and compared with normal donors. Values were normalized to -actin. The relative phosphorylation of the different kinases in PMA-stimulated CD4 lupus T cells was compared with normal-treated T cells arbitrarily considered as 100%. Results shown are the mean percentage of phosphorylation SD of the indicated number of independent experiments.

Article Snippet: The following primary Abs were used: rabbit polyclonal anti-phosphoPKC (Thr638/641), anti-phospho-PKC (Thr538), anti-phospho-PKC (Thr505), anti-phospho-Raf (Ser338), anti-phospho-MEK1/2 (Ser217/221), and anti MEK1/2 used at 1/1000 dilution (Cell Signaling Technology).

Techniques: Phospho-proteomics, Control, Isolation, SDS Page, Membrane, Western Blot