Journal: BMC Veterinary Research

Article Title: Effects of prostaglandin F 2α (PGF 2α ) on cell-death pathways in the bovine corpus luteum (CL)

doi: 10.1186/s12917-019-2167-3

Figure Lengend Snippet: Specific antibodies used for Western immunoblotting and Immmunohistochemistry

Article Snippet: Anti-CASP8 , monoclonal , rabbit , Abcam, ab108333 , 1:1.000 , .

Techniques: Western Blot

Journal: PLoS Genetics

Article Title: Sc65 -Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

doi: 10.1371/journal.pgen.1006002

Figure Lengend Snippet: a) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control were used for IP experiments utilizing a Flag antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 10% SDS-PAGE gel, blotted and probed with antibodies against FLAG and P3H3. The reciprocal interaction of SC65-Flag with P3H3 is confirmed in both experiments. b) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and Sc65KO 3 day-old mice (N = 2) showing significantly decreased levels of P3H3 protein in Sc65KO samples. Densitometric quantification of P3H3 protein normalized to β-actin from the western blot shown above (#p<0.01; *p<0.05; error bars represent SD). All experiments were performed at least 3 times.

Article Snippet: Co-immunoprecipitation of P3H3 and CYPB-HA was carried out the same as LH1-HA except endogenous P3H3 was immunoprecipitated utilizing a polyclonal P3H3 antibody (2.5ug, ProteinTech, Chicago, IL, USA).

Techniques: Stable Transfection, Expressing, Control, SDS Page, Western Blot

Journal: PLoS Genetics

Article Title: Sc65 -Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

doi: 10.1371/journal.pgen.1006002

Figure Lengend Snippet: a) Lysates of 714 mouse embryonic fibroblasts that were transiently transfected with an HA-tagged LH1 expression construct were immuno-precipitated with a HA antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 8% SDS-PAGE gel, blotted and probed with antibodies against HA and P3H3. Negative controls included non-transfected 714 cells incubated with the HA antibody (for non-specific binding of HA antibody, left lanes) and LH1-HA transfected cells incubated with no antibody (for non-specific proteins binding to beads, middle lanes). In both experiments, LH1-HA and P3H3 were found to interact (right lanes). b) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control and transiently transfected with a HA-tagged CYPB were used for IP utilizing an HA antibody. 10% of total input and immuno-precipitates were separated on a 12% SDS-PAGE gel, blotted and probed with antibodies against Flag and HA. The blot detecting SC65-Flag following IP with the HA antibody is shown over-exposed. c) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and Sc65KO 3 day-old mice (N = 2) showing similar content of CYPB protein in Sc65KO and WT samples. All experiments were performed at least 3 times.

Article Snippet: Co-immunoprecipitation of P3H3 and CYPB-HA was carried out the same as LH1-HA except endogenous P3H3 was immunoprecipitated utilizing a polyclonal P3H3 antibody (2.5ug, ProteinTech, Chicago, IL, USA).

Techniques: Transfection, Expressing, Construct, SDS Page, Incubation, Binding Assay, Stable Transfection, Control, Western Blot

Journal: Cell reports

Article Title: CCDC57 Cooperates with Microtubules and Microcephaly Protein CEP63 and Regulates Centriole Duplication and Mitotic Progression.

doi: 10.1016/j.celrep.2020.107630

Figure Lengend Snippet: Figure 6. CCDC57 Is Required for Efficient Ciliogenesis and Centriolar Satellite Distribution (A) Effect of CCDC57 loss on cilium formation. Control and CCDC57-depleted RPE1 cells were serum-starved for 48 h, and percentage of ciliated cells was determined by staining for acetylated-tubulin, glutamylated tubulin, PCM1, and DAPI. (B) Quantification of ciliogenesis experiments. Results shown are the mean of three independent experiments ± SEM (>300 cells per experiment; **p < 0.01). (C) Effect of CCDC57 depletion on cilium length. Cilium length was determined by staining for acetylated-tubulin and DAPI. Results shown are the mean of three independent experiments ± SEM (>100 cells per experiment; ****p < 0.0001). (D) mNeonGreen (mNG)-CCDC57 (1–502) stable expression rescues the ciliogenesis defect of CCDC57-depleted cells. U2OS cells stably expressing mNG, mNG-CCDC57 (1–502), or mNG-CCDC57 (503–916) were fixed 86 h after transfection with control siRNA or CCDC57 siRNA and 24 h serum starvation and were stained for cilium markers. Results shown are the mean of two independent experiments ± SEM (>100 cells per experiment; *p < 0.05; n.s, non-significant). (E) Effect of CCDC57 depletion on centriolar satellite distribution. U2OS cells were fixed 86 h after transfection with control siRNA or CCDC57 siRNA and stained for the indicated proteins. Images represent centrosomes in cells from the same coverslip taken with the same camera settings. (F) Quantification of (E). PCM1 and CEP131 fluorescence intensities at the centrosome were measured from SUM projections, and average means of the levels in control cells were normalized to 1. Data represent mean values from two experiments ± SEM (>50 cells per experiment; **p < 0.01; ****p < 0.0001). (G) Effects of PCM1 depletion on ciliogenesis efficiency of CCDC57-depleted cells. RPE1 cells were transfected with control siRNA, CCDC57 siRNA, or CCDC57/ PCM1 siRNAs for 86 h; serum-starved for the last 24 h; fixed; and stained for cilium markers. (H) Quantification of (G). Results shown are the mean of two independent experiments ± SEM (>100 cells per experiment; **p < 0.01; ***p < 0.001). Scale bars, 10 mm.

Article Snippet: Primary antibodies used for immunoblotting were rabbit anti PCM1 (Proteintech, 19856-1-AP) at 1:500, rabbit anti-beta-actin (Cell Signaling Technology) at 1:10000, mouse anti alpha-tubulin (Sigma, DM1A) at 1:5000, anti-CCDC57 (Sigma HPA023344) at 1:1000, anti-c-Myc (clone 9E10) at 1:500, rabbit anticapsase-3 (Proteintech) at 1:500 and mouse anti-acetylated tubulin (clone 6-11B, 32270, Thermo Fischer) at 1:5000. anti-PCM1 and anti-GFP antibodies were generated and used for immunobloting as previously described (Firat-Karalar et al., 2014).

Techniques: Control, Staining, Expressing, Stable Transfection, Transfection