Journal: BMC Cancer

Article Title: BIM EL is a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

doi: 10.1186/1471-2407-14-27

Figure Lengend Snippet: BSO induces phosphorylation of BIM EL and MCL1 in mitochondria. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NAC or DTT for 12 h. A . The expression and phosphorylation of BIM were determined by immunoblotting. The lower panel indicates a longer exposure of the same blot. B, C . The expression and phosphorylation of BAD, BID, tBID, MCL1, BCLxL and BCL2 were determined by immunoblotting.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): antibodies to the cleaved form of caspase 3 (C-cas3), caspase 9 (C-cas9), poly (ADP-ribose) polymerase (C-PARP); antibodies to normal and phosphorylated forms of MCL1 (MCL1, P-MCL1 at Ser159/Thr163), BCL2 (BCL2, P-BCL2 at Ser70), BIM (BIM, P-BIM at Ser69), JNK (JNK, P-JNK at Thr183/Tyr185), c-JUN (c-JUN, P-c-JUN at Ser63), p38 (p38, P-p38 at Thr180/Tyr182) and ERK1/2 (ERK1/2, P-ERK1/2 at Thr202/Tyr204); antibodies to actin, BAD, BID and BOK.

Techniques: Phospho-proteomics, Expressing, Western Blot

Journal: BMC Cancer

Article Title: BIM EL is a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

doi: 10.1186/1471-2407-14-27

Figure Lengend Snippet: BSO induces the dissociation of phosphorylated BIM EL from MCL1 and the interaction with BAX. A and B , The dissociation of phosphorylated BIM EL and MCL1, and the interaction with BAX were determined by immunoprecipitation and immunoblotting with antibodies to their normal and phosphorylated forms. Values were normalized to actin or IgG(L), respectively and represent relative changes compared with control. A typical result of 3 independent experiments is shown. IP, immunoprecipitation; Ppt, immunoprecipitate; Sup, supernatant.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): antibodies to the cleaved form of caspase 3 (C-cas3), caspase 9 (C-cas9), poly (ADP-ribose) polymerase (C-PARP); antibodies to normal and phosphorylated forms of MCL1 (MCL1, P-MCL1 at Ser159/Thr163), BCL2 (BCL2, P-BCL2 at Ser70), BIM (BIM, P-BIM at Ser69), JNK (JNK, P-JNK at Thr183/Tyr185), c-JUN (c-JUN, P-c-JUN at Ser63), p38 (p38, P-p38 at Thr180/Tyr182) and ERK1/2 (ERK1/2, P-ERK1/2 at Thr202/Tyr204); antibodies to actin, BAD, BID and BOK.

Techniques: Immunoprecipitation, Western Blot, Control

Journal: BMC Cancer

Article Title: BIM EL is a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

doi: 10.1186/1471-2407-14-27

Figure Lengend Snippet: BSO triggers phosphorylation of MCL1and BIM EL via activation of JNK. A , The phosphorylation of JNK, ERK1/2 and p38 was determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NAC or DTT for 12 h. B , The phosphorylation of BIM EL and MCL1, and the cleavage of caspase 3 and PARP, were determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence of SP600125 (10 μM) as a JNK inhibitor, U0126 (2 μM) as an ERK1/2 inhibitor, or SB203580 (10 μM) as a p38 inhibitor, PD035901 (100 nM)as a MEK1/2 inhibitor for 12 h. A typical result of 3 independent experiments is shown.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): antibodies to the cleaved form of caspase 3 (C-cas3), caspase 9 (C-cas9), poly (ADP-ribose) polymerase (C-PARP); antibodies to normal and phosphorylated forms of MCL1 (MCL1, P-MCL1 at Ser159/Thr163), BCL2 (BCL2, P-BCL2 at Ser70), BIM (BIM, P-BIM at Ser69), JNK (JNK, P-JNK at Thr183/Tyr185), c-JUN (c-JUN, P-c-JUN at Ser63), p38 (p38, P-p38 at Thr180/Tyr182) and ERK1/2 (ERK1/2, P-ERK1/2 at Thr202/Tyr204); antibodies to actin, BAD, BID and BOK.

Techniques: Phospho-proteomics, Activation Assay, Western Blot

Journal: BMC Cancer

Article Title: BIM EL is a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

doi: 10.1186/1471-2407-14-27

Figure Lengend Snippet: BSO triggers activation of ASK1 and JNK and induces phosphorylation of BIM EL and MCL1. A , The phosphorylation of ASK1 was determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NAC or DTT for 12 h. B , The phosphorylation of JNK, BIM EL , and MCL1, and cleavage of caspase 3 and PARP, were determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NQDI1 (10 μM) for 12 h. A typical result of 3 independent experiments is shown.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): antibodies to the cleaved form of caspase 3 (C-cas3), caspase 9 (C-cas9), poly (ADP-ribose) polymerase (C-PARP); antibodies to normal and phosphorylated forms of MCL1 (MCL1, P-MCL1 at Ser159/Thr163), BCL2 (BCL2, P-BCL2 at Ser70), BIM (BIM, P-BIM at Ser69), JNK (JNK, P-JNK at Thr183/Tyr185), c-JUN (c-JUN, P-c-JUN at Ser63), p38 (p38, P-p38 at Thr180/Tyr182) and ERK1/2 (ERK1/2, P-ERK1/2 at Thr202/Tyr204); antibodies to actin, BAD, BID and BOK.

Techniques: Activation Assay, Phospho-proteomics, Western Blot

Journal: Cancer letters

Article Title: Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

doi: 10.1016/j.canlet.2013.01.040

Figure Lengend Snippet: Fig. 1. MARCKS and AURKB expression in human colon cancers, Clone A cells and LoVo cells. (A) MARCKS RNA expression was determined in LoVo cells vs Clone A cells. (B) MARCKS protein expression was compared between LoVo cells and Clone A cells. Representative of at least three independent experiments. (C and D) Different RNA expression patrons for MARCKS and AURKB were assessed by QRT-PCR. The expression was compared between three patients classified in Stage I colon carcinoma, tumour (T) vs normal paired colon tissue (N), and two patients with Stage IV liver metastasis tumour (T) vs normal paired liver tissue (N) (P < 0.005 vs normal tissue, P < 0.05 vs normal tissue).

Article Snippet: Primary antibodies against MARCKS (N-19 and M20), P-MARCKS (Ser159/163), a-tubulin (H-300) were purchased from Santa Cruz Biotechnology as well as their secondary antibodies, respectively HRP-conjugated goat or rabbit IgG.

Techniques: Expressing, RNA Expression, Quantitative RT-PCR

Journal: Cancer letters

Article Title: Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

doi: 10.1016/j.canlet.2013.01.040

Figure Lengend Snippet: Fig. 2. RNAi of MARCKS in Clone A cells inhibits cell migration and invasion. (A) Boyden chamber chemotaxis assay was performed using as chemoattractant the conditioned medium (CM) of human hepatic stellate cells (hHSCs-CM). Values are expressed as cells migrated per High Power Field (HPF) and are shown as mean ± SD of four independent experiments. P < 0.05, vs scrambled control cells towards hHSC-CM. (B) Invasion Matrigel assay was performed using hHSC-CM as chemo- attractant. Values are shown as mean ± SD of four independent experiments. P < 0.05, vs scrambled control cells towards hHSC-CM. (C) The reduced chemotaxis of MARCKS-depleted Clone A cells towards hHSC-CM coincides with morphological changes.

Article Snippet: Primary antibodies against MARCKS (N-19 and M20), P-MARCKS (Ser159/163), a-tubulin (H-300) were purchased from Santa Cruz Biotechnology as well as their secondary antibodies, respectively HRP-conjugated goat or rabbit IgG.

Techniques: Migration, Chemotaxis Assay, Control, Matrigel Assay

Journal: Cancer letters

Article Title: Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

doi: 10.1016/j.canlet.2013.01.040

Figure Lengend Snippet: Fig. 3. The actin binding potential of MARCKS limits migration in LoVo cells. (A) MARCKS RNA expression levels were assessed by QRT-PCR in parental LoVo cells transfected with empty vector and compared with LoVo cells transfected with a human MARCKS plasmid (WT), a MARCKS cDNA mutant of the phosphorylation site domain (PSD-m), and a MARCKS myristoylated cDNA mutant (Myr-m). (B) Western blot analysis demonstrates the transfection efficiency. (C) Mutational analysis was performed followed by a Boyden chamber chemotaxis assay using hHSC-CM as chemoattractant. Values are shown as mean ± SD of two independent experiments. P < 0.005, vs empty vector, #P < 0.05, vs MARCKS-WT. Wild type MARCKS transfected LoVo cells changed cell morphology in comparison to control cells when hHSC-CM was used as chemoattractant. (D) Invasion Matrigel assay was performed using hHSC-CM as chemoattractant. Values are shown as mean ± SD of two independent experiments, P < 0.05, vs empty vector.

Article Snippet: Primary antibodies against MARCKS (N-19 and M20), P-MARCKS (Ser159/163), a-tubulin (H-300) were purchased from Santa Cruz Biotechnology as well as their secondary antibodies, respectively HRP-conjugated goat or rabbit IgG.

Techniques: Binding Assay, Migration, RNA Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Mutagenesis, Phospho-proteomics, Western Blot, Chemotaxis Assay, Comparison, Control, Matrigel Assay

Journal: Cancer letters

Article Title: Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

doi: 10.1016/j.canlet.2013.01.040

Figure Lengend Snippet: Fig. 4. Changing MARCKS protein expression affects cell proliferation. Interplay between MARCKS and AURKB. (A) RNAi against MARCKS in Clone A cells coincides with a reduction in cell proliferation as measured by Methyl-[ 3H]-thymidine incorporation into DNA (n = 3, P < 0.05 vs scrambled control). MARCKS WT cDNA-transfected LoVo cells were trypsinized, stained and cell number was counted. MARCKS re-expression inhibits cell proliferation (n = 3, in duplicate, P < 0.05, vs vector control cells). (B) MARCKS- depleted Clone A cells and MARCKS WT cDNA-transfected LoVo cells affect AURKB protein expression. (C) AURKB-depletion in Clone A cells does not affect MARCKS protein expression. Western blot analysis demonstrates the effect of RNAi against AURKB in Clone A cells and LoVo cells (a representative picture and histogram is shown of two independent experiments, P < 0.005).

Article Snippet: Primary antibodies against MARCKS (N-19 and M20), P-MARCKS (Ser159/163), a-tubulin (H-300) were purchased from Santa Cruz Biotechnology as well as their secondary antibodies, respectively HRP-conjugated goat or rabbit IgG.

Techniques: Expressing, Control, Transfection, Staining, Plasmid Preparation, Western Blot

Journal: Cancer letters

Article Title: Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

doi: 10.1016/j.canlet.2013.01.040

Figure Lengend Snippet: Fig. 5. Down-regulation of MARCKS expression inhibits migration and invasion of CT26 in vitro . (A) Western blot analysis showed the transfection efficiency of CT26 cells stable shRNA-depleted for MARCKS with different shRNA MARCKS (constructs 1–3), shRNA-C is empty vector and shRNA-SC is the scrambled control. (B) A Boyden chamber chemotaxis assay was used with mHSC-CM as chemoattractant. Values are shown as mean ± SD of three independent experiments. P < 0.005 vs shRNA scrambled control. (C) Invasion Matrigel assays were performed using mHSC-CM as chemoattractant. Values are shown as mean ± SD of three independent experi- ments. Invasion was reduced when mHSC-CM was used as chemoattractant. P < 0.005 vs shRNA scrambled control cells.

Article Snippet: Primary antibodies against MARCKS (N-19 and M20), P-MARCKS (Ser159/163), a-tubulin (H-300) were purchased from Santa Cruz Biotechnology as well as their secondary antibodies, respectively HRP-conjugated goat or rabbit IgG.

Techniques: Expressing, Migration, In Vitro, Western Blot, Transfection, shRNA, Construct, Plasmid Preparation, Control, Chemotaxis Assay

Journal: Cancer letters

Article Title: Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

doi: 10.1016/j.canlet.2013.01.040

Figure Lengend Snippet: Fig. 6. Down-regulation of MARCKS protein delays duration time of mitosis in CT26 cells. (A) Phase contrast imaging shows that shRNA MARCKS-depleted cells obtain a mesenchymal-like phenotype during interphase in contrast to the epithelial-like morphology observed in scrambled control cells. (B) A down-regulation in MARCKS protein expression inhibits cell cycle as was measured by MTT assay (two independent experiments performed each in 6-fold). Values are shown as mean ± SD, P < 0.005 vs scrambled control. (C) A Montage of live cell imaging of shRNA scrambled control cells. Numbers in images indicate time (in minutes) since entering in mitosis. (D) Snapshots of a montage shows that shRNA MARCKS-depleted cells are marked with a delay in DTM that coincides with a morphological aberrant phenotype of the two daughter cells. (E) shRNA MARCKS stable transfected cells show a significant increase in the duration time of mitosis (DTM) when compared to scrambled control cells (sum of DTM, values are shown as mean ± SD, P < 0.005 vs scrambled control). Scrambled control cells have an average DTM of 160 min (marked as t time = 100%) whereas 51.2% of MARCKS depleted cells have a delay in DTM (marked as >t) (n = 3). (F) Representative Western blot analysis shows the absence of MARCKS protein expression in stable transfected shRNA MARCKS-depleted cells (n = at least 3).

Article Snippet: Primary antibodies against MARCKS (N-19 and M20), P-MARCKS (Ser159/163), a-tubulin (H-300) were purchased from Santa Cruz Biotechnology as well as their secondary antibodies, respectively HRP-conjugated goat or rabbit IgG.

Techniques: Imaging, shRNA, Control, Expressing, MTT Assay, Live Cell Imaging, Transfection, Western Blot

Journal: Cancer letters

Article Title: Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

doi: 10.1016/j.canlet.2013.01.040

Figure Lengend Snippet: Fig. 7. The effect of MARCKS-depletion in CT26 cells attenuates tumour progression in vivo . (A) Representative Western blot analysis shows the stable transfection efficiency of shRNA against MARCKS in CT26 cells. (B) Photographs of representative liver after performing a colorectal liver metastasis assay in vivo . In both conditions, tumour formation was observed in the intrasplenic injection site, whereas hepatic metastases were detectable in SCRNA CT26 tumour-challenged mice with few or no tumour formation in MARCKS-depleted CT26 cells tumour-challenged mice. (C) Graphs show the body–liver weight (%) and demonstrates a significant induction of CT26 and MARCKS-depleted CT26 tumour-challenged mice vs sham-operated (P < 0.05, vs sham-operated mice). Tumour-challenged mice with shRNA MARCKS-depleted CT26 cells showed few or no metastases in comparison to CT26 tumour-challenged mice (P < 0.005 vs shRNA SC). (D) Haematoxylin-eosin staining confirmed the presence of hepatic tumours in CT26 tumour-challenged mice.

Article Snippet: Primary antibodies against MARCKS (N-19 and M20), P-MARCKS (Ser159/163), a-tubulin (H-300) were purchased from Santa Cruz Biotechnology as well as their secondary antibodies, respectively HRP-conjugated goat or rabbit IgG.

Techniques: In Vivo, Western Blot, Stable Transfection, shRNA, Injection, Comparison, Staining

Journal: Reproduction (Cambridge, England)

Article Title: Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs.

doi: 10.1530/rep.1.00794

Figure Lengend Snippet: Figure 1 Localization of MARCKS at var- ious stages of in vivo fertilization. Eggs were labeled with anti-MARKCS goat polyclonal IgG (1:50) and Hoechst (1 mg/ml). Localization of the antibodies was imaged using a donkey anti-goat IgG Cy secondary antibody (1:300) and CLSM. (A, E and I) Control, second anti- body only; (B, F and J) unfertilized egg; (C, G and K) egg at the SB stage; (D, H and L) egg at the PBII stage. (A–D) Light microscopy; (E–L) MARCKS (green) and chromosomes (blue); sperm DNA, yellow arrow; egg DNA, white arrow. (I–L) Cross-section at the equatorial plane of the egg is shown, depicting the cortical area at £ 4 magnification. At least three independent experiments were per- formed (three to four eggs for each group in each experimental day). Each image was taken at the equatorial plane of the egg. Scale bar, 10 mm.

Article Snippet: Anti-MARCKS goat polyclonal immunoglobulin G (IgG) (N-19, sc-6454; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); MARCKS peptide (N-19; Santa Cruz); anti-phosphorylated-MARCKS rabbit polyclonal IgG (Ser159/163-R, sc-12971-R; Santa Cruz); anti-CaM rabbit polyclonal IgG (FL-149, sc-5537; Santa Cruz); anti-actin rabbit polyclonal IgG (A-5060; Sigma).

Techniques: In Vivo, Labeling, Control, Light Microscopy

Journal: Reproduction (Cambridge, England)

Article Title: Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs.

doi: 10.1530/rep.1.00794

Figure Lengend Snippet: Figure 2 MARCKS translocation follow- ing activation by various activators. Sub- cellular localization of MARCKS was visualized after exposing MII eggs to PKC activators: 30 ng/ml TPA or 2 mM iono- mycin for 5 min; or 20 mg/ml OAG for 3 min. Eggs were fixed at the MII stage (A and E, A0 and E0, A00 and E00); after a 3–5 min incubation in the presence of an activator (B and F, B0 and F0, B00 and F00); after a 3-5 min incubation in the pre- sence of an activator followed by an additional incubation in fresh medium lacking the activator (10 or 15 min (C and G, C0 and G0, D and H, D0 and H0); 2 or 12 min (C00 and G00, D00 and H00). (A–H) TPA-treated eggs; (A0 –H0) ionomy- cin-treated eggs; (A00 –H00) OAG-treated eggs. Eggs were labeled with anti- MARKCS goat polyclonal IgG (1:50) and Hoechst (1 mg/ml). Localization of the antibodies was imaged using donkey anti-goat IgG Cy secondary antibody (1:300) and CLSM: MARCKS, green; chromosomes, blue. For localization of MARCKS, eggs were scanned using the CLSM Z-axis to visualize sections through their equatorial plane and through the cortex. To allow a better observation of the egg cortex, the eggs, already visualized at the equatorial plane, were pressed between the slide and the coverslip and scanned. The images at the equatorial plan and the cortex, are not necessarily of the same egg: images at the equatorial plane of the egg (A–D, A0 –D0, A00 –D00); images at the cortex of the egg (E–H, E0 –H0, E00 –H00). At least three independent experiments were performed (three to four eggs for each group in each experimental day). Scale bar, 10 mm.

Article Snippet: Anti-MARCKS goat polyclonal immunoglobulin G (IgG) (N-19, sc-6454; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); MARCKS peptide (N-19; Santa Cruz); anti-phosphorylated-MARCKS rabbit polyclonal IgG (Ser159/163-R, sc-12971-R; Santa Cruz); anti-CaM rabbit polyclonal IgG (FL-149, sc-5537; Santa Cruz); anti-actin rabbit polyclonal IgG (A-5060; Sigma).

Techniques: Translocation Assay, Activation Assay, Incubation, Labeling

Journal: Reproduction (Cambridge, England)

Article Title: Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs.

doi: 10.1530/rep.1.00794

Figure Lengend Snippet: Figure 3 Phosphorylation of MARCKS after activation by TPA. Eggs, before or after parthenogenetic activation by TPA (50 ng/ml) were pooled, lysed and the proteins were separated on SDS-PAGE (400 eggs per lane). The proteins were immunoblotted with anti p- MARKCS rabbit polyclonal IgG (1:200; A) and anti-actin rabbit polyclonal IgG (1:250; B). Peroxidase-conjugated donkey anti-rabbit IgG secondary antibody was used (1:5000) followed by an ECL detec- tion system. The arrow points to the phosphorylated MARCKS protein at 80 kDa as calculated from the migration of protein standards with known molecular masses. At least three independent experiments were performed.

Article Snippet: Anti-MARCKS goat polyclonal immunoglobulin G (IgG) (N-19, sc-6454; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); MARCKS peptide (N-19; Santa Cruz); anti-phosphorylated-MARCKS rabbit polyclonal IgG (Ser159/163-R, sc-12971-R; Santa Cruz); anti-CaM rabbit polyclonal IgG (FL-149, sc-5537; Santa Cruz); anti-actin rabbit polyclonal IgG (A-5060; Sigma).

Techniques: Phospho-proteomics, Activation Assay, SDS Page, Migration

Journal: Reproduction (Cambridge, England)

Article Title: Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs.

doi: 10.1530/rep.1.00794

Figure Lengend Snippet: Figure 4 Effect of PKC inhibitor on MARCKS translocation. Eggs were fixed at the MII stage (A and D); after a 5-min incubation in the pre- sence of 30 ng/ml TPA followed by an additional 15-min incubation in fresh medium lacking the activator (B and E); after a 30-min incubation in the presence of 35 mM myrPKCc followed by a 5-min incubation in the presence of 30 ng/ml TPA and 35 mM myrPKCc, followed by 15-min incubation in medium containing 35 mM myrPKCc but without TPA (C and F). Eggs were labeled with anti- MARKCS goat polyclonal IgG (1:50) and Hoechst (1 mg/ml). Localiz- ation of the antibodies was imaged using donkey anti-goat IgG (Cy) secondary antibody (1:300) and CLSM. (A–C) Light microscopy and chromosomes; (D–F) MARCKS. Images were taken at the equatorial plane of the egg. At least three independent experiments were per- formed (three to four eggs for each group in each experimental day. Scale bar, 10 mm.

Article Snippet: Anti-MARCKS goat polyclonal immunoglobulin G (IgG) (N-19, sc-6454; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); MARCKS peptide (N-19; Santa Cruz); anti-phosphorylated-MARCKS rabbit polyclonal IgG (Ser159/163-R, sc-12971-R; Santa Cruz); anti-CaM rabbit polyclonal IgG (FL-149, sc-5537; Santa Cruz); anti-actin rabbit polyclonal IgG (A-5060; Sigma).

Techniques: Translocation Assay, Incubation, Labeling, Light Microscopy

Journal: Reproduction (Cambridge, England)

Article Title: Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs.

doi: 10.1530/rep.1.00794

Figure Lengend Snippet: Figure 5 Expression and localization of CaM in the egg. (A) Western blot analysis. Samples of 200 MII eggs were pooled, lysed and the proteins were revealed by SDS-PAGE analysis. The proteins were immunoblotted with anti-CaM rabbit polyclonal IgG (1:200). Second- ary antibody, peroxidase-conjugated goat anti-rabbit IgG (1:5000) was followed by an ECL detection system. The arrow points to the actin at 17 kDa as calculated from the migration of protein standards with known molecular masses. At least three independent exper- iments were performed. (B) Immunofluorescence localization. Eggs were fixed at the MII stage and labeled with anti-CaM rabbit polyclo- nal IgG (1:50) and Hoechst (1 mg/ml). Localization of the antibodies was imaged using donkey anti-rabbit IgG Cy secondary antibody (1:300) and CLSM. A representative egg is presented. Left panel, light microscopy and chromosomes; right panel, CaM. At least three inde- pendent experiments were performed (three to four eggs for each group in each experimental day). Scale bar, 10 mm. (C) Association of MARCKS with CaM during egg activation. One thousand unfertilized MII eggs or ionomycin (2 mM)- activated eggs were lysed. The lysis buffer (control) and eggs lysates were immunoprecipitated with anti- CaM rabbit polyclonal IgG (‘IP-CaM’ on figure). Proteins were resolved by SDS-PAGE analysis and transferred onto nitrocellulose membrane. The blots were probed with anti-MARKCS goat polyclo- nal IgG (1:50; ‘Blot-MARCKS’ on figure) and revealed by the ECL detection system. The arrow points to MARCKS at 80 kDa as calcu- lated from the migration of protein standards with known molecular masses. The result of a representative experiment is presented. At least three independent experiments were performed.

Article Snippet: Anti-MARCKS goat polyclonal immunoglobulin G (IgG) (N-19, sc-6454; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); MARCKS peptide (N-19; Santa Cruz); anti-phosphorylated-MARCKS rabbit polyclonal IgG (Ser159/163-R, sc-12971-R; Santa Cruz); anti-CaM rabbit polyclonal IgG (FL-149, sc-5537; Santa Cruz); anti-actin rabbit polyclonal IgG (A-5060; Sigma).

Techniques: Expressing, Western Blot, SDS Page, Migration, Labeling, Light Microscopy, Activation Assay, Lysis, Control, Immunoprecipitation, Membrane

Journal: Reproduction (Cambridge, England)

Article Title: Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs.

doi: 10.1530/rep.1.00794

Figure Lengend Snippet: Figure 6 Effect of a CaM inhibitor on MARCKS translocation. Eggs fixed at the MII stage (A and D); after a 5-min incubation in the pre- sence of 2 mM ionomycin followed by an additional 15-min incu- bation in fresh medium lacking the activator (B and E); after a 30-min incubation in the presence of 25 mM W7 followed by activation with 2 mM ionomycin for 5 min in the presence of 25 mM W7, followed by a 15-min incubation in TH medium that contains 25 mM W7 without ionomycin (C and F). Eggs were labeled with anti-MARKCS goat polyclonal IgG (1:50) and Hoechst (1 mg/ml). Light microscopy and chromosomes (blue, A–C); MARCKS (green, D–F). Images were taken at the equatorial plane of the eggs. At least three independent experiments were performed (three to four eggs for each group in each experimental day). Scale bar, 10 mm.

Article Snippet: Anti-MARCKS goat polyclonal immunoglobulin G (IgG) (N-19, sc-6454; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); MARCKS peptide (N-19; Santa Cruz); anti-phosphorylated-MARCKS rabbit polyclonal IgG (Ser159/163-R, sc-12971-R; Santa Cruz); anti-CaM rabbit polyclonal IgG (FL-149, sc-5537; Santa Cruz); anti-actin rabbit polyclonal IgG (A-5060; Sigma).

Techniques: Translocation Assay, Incubation, Activation Assay, Labeling, Light Microscopy