Expression of CK1δ and ε in naïve and regenerating retina. Similar levels of CK1δ and ε expression in retinal lysates of untreated animals (C) and rats subjected to optic nerve cut (ONC), lens injury (LI) or ONC+LI were detected by western blot analysis using the CK1δ specific antibody 128A and the CK1ε specific antibody #610446. Detection of β-actin on stripped membranes verified loading of same protein amounts.

(A) Expression of CK1δ and ε in PC12 cells. CK1δ and ε expression levels were detected in protein lysates of untreated and PC12 cells exposed to NGF for 1–48 h by western blot analyses using CK1δ (128A) and CK1ε (#610446) specific antibodies. β-actin (detected on stripped membranes) served as loading control. (B) Densitometric evaluation of western blot results shown in (A). Effects of NGF treatment are significant at * p<0.05 and ** p<0.01 when compared to untreated control cells. (C, D) Detection of CK1δ expression in PC12 cells after exposure to NGF for 48 h using antibodies for CK1δ (serum NC10; red) and βIII-tubulin (MAB1637; green). CK1δ positive staining was observed in cell bodies, in neurites and in growth cones (C). Neurite and growth cone are presented at higher magnification in (D). Scale bar for C: 20 µm. Scale bar for D: 2.5 µm. (E, F) CK1ε immunostaining in PC12 cells after exposure to NGF for 48 h using antibodies for CK1ε (serum 712; red) and βIII-tubulin (MAB1637; green). CK1ε was detected in the cytoplasm, in neurites and in the growth cones. A magnification of a neurite and growth cone is presented in (F). Scale bar for E: 50 µm. Scale bar for F: 2 µm.

Equal protein amounts of lysates from untreated PC12 cells or PC12 cells exposed to NGF for 24 h were loaded onto a 1 ml Resource-Q column for ion exchange chromatography and eluted with a linear gradient of increasing NaCl concentration (represented as a solid diagonal line). Kinase activity was determined in each single fraction using the GST-CK1δ^305–375 fusion protein (FP1006) as substrate. Quantification of phosphate incorporation was measured by Cherenkov counting. The major kinase activity eluted at 340–410 mM NaCl in extracts prepared before (open squares) or 24 h (closed triangles) after the induction of differentiation processes of PC12 cells. Data in Fig. 5 show one representative experiment of two.

(A) LI stimulated RGCs were cultured for 24 h and then exposed to vehicle or different concentrations of the CK1-specific inhibitors IC261 (0.5, 1.5 and 50 µM) or CKI-7 (50 and 200 µM). The average neurite length of RGCs was determined after 24 h (first control) and 48 h, respectively. Treatment effects: ***p<0.001. (B) The number of RGCs per well in all groups was not affected by any treatment described in (A). (C) LI stimulated RGCs were cultured for 48 h and subsequently monitored in a flow-through chamber for live cell imaging. In the presence of vehicle or inhibitors IC261 (50 µM) or D4476 (5 µM) cells were recorded for 4 h. Retraction of the outgrown neurite after exposure to IC261 and D4476 is shown at 0, 120 and 240 min. Arrowheads indicate the position of the cell body, arrows the position of the growth cone. Scale bar: 50 µm. (D) Quantification of neurite retraction of RGCs over time exposed to vehicle (control, closed squares) or inhibitors IC261 (50 µM, open triangles) or D4476 (5 µM, open squares) specifically inhibiting CK1δ and ε.

(A) Immunofluorescence staining and phase contrast image of the neurite growth cone of a RGC using the CK1δ specific monoclonal antibody 128A (red) and a βIII-tubulin specific monoclonal antibody (RB-9249-P0; green). Epifluorescence microscopy of RGCs revealed that CK1δ is located in granular particles aligned at microtubules all over the growth cone. Distribution of CK1δ in the soma is shown in the small vertical image panel (δ: CK1δ, tub: βIII-tubulin, m: merged). Scale bars: 10 µm. (B) Co-staining of CK1ε (serum 712; red) and βIII-tubulin (TUJ-1; green) in RGCs revealed a similar expression pattern as shown for CK1δ in (A). Distribution of CK1ε in the soma is shown in the small vertical image panel (ε: CK1ε, tub: βIII-tubulin, m: merged). Scale bars: 10 µm.

(A) Kinase activity in PC12 cells. Equal amounts of protein lysates from PC12 cells either untreated (control) or exposed to NGF for 24 h were loaded onto a 1 ml Resource-Q column for ion exchange chromatography and eluted with a linear gradient of increasing NaCl concentration (represented as a solid diagonal line). Fractions were collected and the CK1δ/ε specific kinase activity was determined in each single fraction as described in detail in Material and Methods. Kinase activities (CPM: counts per minute) are represented by open squares (control) or closed triangles (24 h after NGF stimulation). (B) The kinase activity in peak fraction 21 of untreated (control) and NGF stimulated PC12 cells (24 h) was significantly reduced by IC261 specifically inhibiting CK1δ and ε in the micromolar range. Phosphate incorporation into GST-p53^1–64 (FP267) was quantified by Cherenkov counting. (C) Kinase activity in rat retina. Equal amounts of protein lysates from rat retinal tissue either untreated or treated with ONC+LI were loaded onto a 1 ml Resource-Q column for ion exchange chromatography and eluted with a linear gradient of increasing NaCl concentration (represented as a solid diagonal line). Fractions were collected and the CK1δ/ε specific kinase activity was determined in each single fraction as described in detail in Material and Methods. Kinase activities (CPM: counts per minute) are represented by open squares (control) or closed triangles (ONC+LI). (D) The kinase activity in peak fraction 38 of extracts prepared from ONC+LI treated animals was significantly reduced by the CK1δ/ε specific inhibitor IC261. Phosphate incorporation into GST-p53^1–64 (FP267) was quantified by Cherenkov counting.

(A) PC12 cells were cultured for 3 days without NGF (control) and in the presence of NGF+vehicle or NGF+CKI-7 (50 and 200 µM) or IC261 (0.5, 1.5 and 50 µM). In a concentration dependent manner the average neurite length of NGF stimulated PC12 cells was markedly reduced in the presence of the CK1-specific inhibitors CKI-7 or IC261. Treatment effects: **p<0.01 and ***p<0.001 compared to cells exposed to NGF+vehicle. (B, C) LI stimulated RGCs were cultured in the absence (control) or presence of CKI-7 (B; 50, 200 and 800 µM) or IC261 (C; 0.5, 1.5 and 50 µM). Neurite outgrowth was compromised in a concentration dependent manner. The survival of RGCs was only affected in the presence of CKI-7 at 800 µM. Treatment effects compared to control groups: *p<0.05, **p<0.01, ***p<0.001.