Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: In vivo detection of promoter occupancy by the E2F and pRB family using chromatin immunoprecipitations. (A) Outline of the in vivo cross-linking and chromatin immunoprecipitation (IP) protocol. (B) Promoters examined in this study. Solid ovals represent E2F-binding sites on the basis of previous studies; small arrows indicate primers used in PCR amplification reactions. Large arrows represent published major transcription start site. (C) Chromatin immunoprecipitations from asynchronously growing T98G cells. Input corresponds to PCR reactions containing 0.5% of total amount of chromatin used in immunoprecipitation reactions. Mock immunoprecipitations performed with irrelevant, control antibodies (anti-T Antigen pAb101) and control reactions lacking antibodies (No Ab) are shown. Amplified products were detected by autoradiography.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: In Vivo, Chromatin Immunoprecipitation, Binding Assay, Amplification, Immunoprecipitation, Autoradiography

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Cell cycle synchronization and gene expression analysis. (A) FACS analysis of T98G cells that were rendered quiescent by serum starvation (0 hr) and were subsequently restimulated with serum for the indicated lengths of time and allowed to enter the cell cycle. DNA content (propidium iodide intensity) is plotted vs. cell number. (B) Northern blot analysis of expression of the indicated genes during the synchronization experiment in A. To ensure equal loading of RNA, each blot was subsequently stripped and rehybridized with an ARPP P0 gene fragment, and a representative filter is shown. (C) Gel mobility shift analysis of E2F and pRB complexes. Whole cell extracts at each stage of the cell cycle (indicated at top) were incubated with a labeled probe containing an E2F site and each of the designated antibodies. The positions of E2F-pRB/p107/p130 complexes and free E2F (E2F) are shown at right. Each of these complexes was abrogated by a specific competitor oligonucleotide (data not shown). Asynchronous (AS) samples were prepared from cells entering a second cell cycle (with roughly equal percentages of G1, S, and G2 cells). Lanes marked − and control contained no antibody and 12CA5 (anti-flu hemagglutinin antibody), respectively. α-E2F-4 lanes contain two different monoclonal antibodies specific for E2F-4 (WUF3 and LLF4, respectively).

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Expressing, Northern Blot, Mobility Shift, Incubation, Labeling

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: The binding of E2F family of proteins changes during the cell cycle. Chromatin immunoprecipitations were performed with antibodies specific for individual E2F family members as indicated, and the resulting immunoprecipitates were amplified with primer pairs corresponding to the genes shown. G0, serum-starved cells; early G1, late G1, G1/S, and S-phase cells were stimulated with serum 4–8, 16, 20, and 24 hr, respectively. Antibodies tested in each case are listed in Materials and Methods.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Binding Assay, Amplification

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Antibodies against each pRB family member immunoprecipitate their chromatin-bound targets. (A) Schematic representing the position of chromatin in gradients relative to free DNA and protein under conditions in which cells were either fixed with formaldehyde (+ cross-linker) or left untreated (− cross-linker). Brackets indicate chromatin fractions selected for further analysis in B. (B) Fractions in A were immunoprecipitated with anti-pRB, anti-p107, and p130 antibodies as indicated and the resulting precipitates were probed with the same antibodies as indicated. (C) p107 and p130 are detected at similar levels in T98G cells entering a second cell cycle (32-hr time point in Fig. ​Fig.2).2). Chromatin immunoprecipitations were performed with the indicated antibodies, and E2F-1, cyclin A, and actin promoter fragments were amplified as described in the legend to Fig. ​Fig.1.1. Distinct monoclonal and polyclonal anti-pRB antibodies were tested as shown.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Immunoprecipitation, Amplification

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Histone acetylation levels of E2F-responsive genes change during the cell cycle. (A) Chromatin was prepared from synchronized T98G cells (as described in Fig. ​Fig.2A)2A) and immunoprecipitated with antibodies specific for acetylated histone H3 and acetylated H4 as described in the legend to Fig. ​Fig.1.1. Cell cycle stages were identical to those described in Fig. ​Fig.3.3. Parallel immunoprecipitations without antibody or with an irrelevant antibody control failed to yield detectable signals after an equivalent autoradiographic exposure (data not shown). (B) Data in A were quantitated by PhosphorImager analysis and normalized vs. input levels. The y-axis indicates fold acetylation (in arbitrary units). (Light-colored bars) acetylated H3; (dark bars) acetylated H4.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Immunoprecipitation

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Model for in vivo occupancy by E2F and pRB family members during cell cycle progression. (A) In vivo occupation of E2F-1, Cdc25A, cyclin A, Cdc6, and p107 promoters by the E2F and pRB family during cell cycle progression and transcriptional consequences. (B) In vivo binding of B-myb promoter. In A and B, promoter binding by E2F-4 correlates with transcriptional repression, whereas binding by E2F-1 and E2F-3 occurs at a time when each promoter is activated. However, the B-myb promoter is transcriptionally active after E2F is no longer bound, consistent with Zwicker et al. (1996). (D) HDAC activity; (H) nucleosomes; (Ac) acetylated histones; (HAT) histone acetyltransferase. These promoters are likely to be regulated by additional trans-activator proteins proximal to E2F, including Sp1. Recruitment of deacetylase and HAT activities could occur by direct interactions with E2F complexes or could require additional factors (X) yet to be defined.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: In Vivo, Binding Assay, Activity Assay, Histone Deacetylase Assay

Journal: Cancer cell

Article Title: Long-term breast cancer response to CDK4/6 inhibition defined by TP53-mediated geroconversion

doi: 10.1016/j.ccell.2024.09.009

Figure Lengend Snippet: (A) GESA plot with normalized enrichment score (NES) of REACTOME gene sets. NES was analyzed between MCF7 and p53KO treated with 50 nM abemaciclib at day21. REACTOME-E2F-mediated-regulation-of-DNA-replication (top), REACTOME-Transcription-of-E2F-targets-under-negative-control-by-DREAM-complex (middle) and REACTOME-Transcription-of-E2F-targets-under-negative-control-byp107 RBL1-and-p130 RBL2-in-complex-with-HDAC1 (bottom). (B) Immunoblotting of DREAM complex components with 50 nM abemaciclib. (C) Cells were treated with 100 nM abemaciclib for 48 h. Lysates were immunoprecipitated with p130 and IgG antibodies. (D) Cells were treated with 7 days of abamacilib and tested by ChIP followed by real-time PCR. Bar showed technical duplicate. Unpaired, Student’s t test. (E) Western blot results of MCF7 cells and p53KO transduced with doxycycline (dox)-inducible HA-tagged p21. The cells were treated with 50 nM abemaciclib or DMSO +/− dox for 48 h. (F) Dox-inducible HA-p21 MCF7 and p53KO cells were treated with 100 nM abemaciclib and +/− dox for 72 h. Lysates were immunoprecipitated with E2F4 and IgG antibodies. (G) Time course change of the proportion of G1 phase after drug withdrawal. FUCCI labeled MCF7 were treated with 50 nM abemaciclib and FUCCI labeled p53KO cells were treated with or without dox and with 50 nM abemaciclib for 4 days. The cells were cultured without abemaciclib for 62 h under time-lapse imaging. Two-way ANOVA, Tukey’s. (H) Cell viability treated with 50 nM abemaciclib or DMSO +/− dox. Data are means ± SEM of four replicates. Two-way ANOVA, Tukey’s. (I) Flow cytometry analysis showing the percentage of SA-β-Gal positive cells. Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (J) Colony formation assay. Cells were treated with 50 nM abemaciclib ± dox for 11 days and reseeded without drug. (K) Cell viability of p130KO in dox inducible HA-p21 cells. The cells were treated with 50 nM abemaciclib or DMSO +/− dox. Data are means ± SEM of six replicates. Two-way ANOVA, Tukey’s. See also and .

Article Snippet: E2F4 (IP) , Santa Cruz Biotechnology , sc-398543.

Techniques: Negative Control, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transduction, Labeling, Cell Culture, Imaging, Flow Cytometry, Colony Assay

Journal: Molecular and Cellular Biology

Article Title: Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

doi: 10.1128/mcb.00872-09

Figure Lengend Snippet: FIG. 6. C/EBP regulates the expression of E2F family genes in E-treated uterine epithelial cells. (A) LE cells were isolated from uteri of ovariectomized WT or C/EBP/ mice following treatment with E for 0, 12, and 15 h. (a to c) Expression levels of E2F1 (a), E2F2 (b), and E2F3 (c) were determined by real-time PCR. The fold changes indicate mRNA expression levels of the E2F genes relative to those of E-treated WT cells at 0 h. (d) Relative steady-state levels of mRNAs for E2Fs 1, 2, and 3 in primary LE cells obtained from uteri treated with E for 15 h are shown following normalization with the 36B4 mRNA level. Statistically significant differences (P 0.05) are indicated by asterisks. (B) Uteri from WT and C/EBP-null mice treated with E for 15 h were collected, and sections were subjected to IHC analysis using anti-E2F3 antibody. Positive staining for E2F3 is indicated in red, and DAPI-stained nuclei are shown in blue. (C) ChIP analysis. LE cells were isolated from uteri of ovariectomized WT mice following treatment with E for 12 h. ChIP was performed as described in Materials and Methods using antibodies against C/EBP, RNA polymerase II (RNAP II), and rabbit IgG. Relative levels of recruitment at various sites on the E2F3 promoter were determined by real-time PCR and normalized to input DNA and RNA polymerase II values.

Article Snippet: Sections were incubated with the following primary antibodies diluted in blocking solution (0.25% bovine serum albumin [BSA], 0.3% Triton X-100, sterile PBS) overnight at 4°C: BrdU and Ki67 (BD Pharmingen), phospho-Ser10 histone H3 (Upstate Biotechnology); cyclin E, cyclin A, and Rad18 (Abcam); cyclin D1 (LabVision NeoMarkers); E2F3 (Santa Cruz Biotechnology); p27 (BD Transduction Laboratories); and phospho(Ser1981)-ATM, phospho-(Ser139)-H2AX, caspase 3, and cleaved caspase 3 (Cell Signaling Technology).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Staining

Journal: Molecular and Cellular Biology

Article Title: Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

doi: 10.1128/mcb.00872-09

Figure Lengend Snippet: FIG. 10. Molecular pathways regulated by C/EBP during E-in- duced proliferation of uterine epithelial cells. In mouse uterine epi- thelial cells, the expression of C/EBP is stimulated by E and opposed by simultaneous treatment with P. C/EBP controls the entry of E- stimulated epithelial cells into the S phase of the cell cycle by upregu- lating the expression of E2F3 and cyclin E. It also promotes the nuclear localization of cyclin E, which enables the formation of an active cyclin E-cdk2 complex critical for DNA replication. The lack of C/EBP allows the nuclear accumulation of p27, which contributes to the cell cycle arrest. In E-stimulated C/EBP-null uterine epithelial cells, stalled DNA replication activates a DNA damage response path- way involving ATM/ATR, Chk1/Chk2, Rad18, and p53. Activated p53 maintains the block in DNA replication by enhancing the synthesis of the cell cycle inhibitor p21 and also promotes the removal of damaged cells via caspase-dependent apoptosis.

Article Snippet: Sections were incubated with the following primary antibodies diluted in blocking solution (0.25% bovine serum albumin [BSA], 0.3% Triton X-100, sterile PBS) overnight at 4°C: BrdU and Ki67 (BD Pharmingen), phospho-Ser10 histone H3 (Upstate Biotechnology); cyclin E, cyclin A, and Rad18 (Abcam); cyclin D1 (LabVision NeoMarkers); E2F3 (Santa Cruz Biotechnology); p27 (BD Transduction Laboratories); and phospho(Ser1981)-ATM, phospho-(Ser139)-H2AX, caspase 3, and cleaved caspase 3 (Cell Signaling Technology).

Techniques: Expressing, Blocking Assay

Journal: Nature cell biology

Article Title: Canonical and atypical E2Fs regulate the mammalian endocycle.

doi: 10.1038/ncb2595

Figure Lengend Snippet: Figure 2 E2f7 and E2f8 promote TGC endocycles. (a) NanoString analysis of TGC-specific E2f4–6 (left panel) and E2f7/E2f8 (right panel) expression in laser-capture-microdissected wild-type cells; n = 2 placentae analysed per time point. (b) Immunohistochemistry demonstrating E2F7 (top left) and E2F8 (bottom left) expression in wild-type E10.5 TGCs but not mutant controls (right panels). The arrows point to selected TGCs. (c) Representative H&E-stained sections of E10.5 control and E2f7−/−;E2f8−/−(78dko) placentae. Inset, higher magnification of the outlined area, showing a 78dko TGC in metaphase. (d) Feulgen quantification of genome ploidy in E10.5 TGCs; n = 3 placentae analysed per genetic group. (e–g) Immunostaining and quantification of S and M phase proteins in E10.5 control and 78dko TGCs. The arrows point to TGCs. n = 3 placentae analysed per genetic group. (h) Co-immunofluorescence microscopy showing E10.5 78dko TGCs in anaphase (left, P-H3) and metaphase (right, PL-1). DAPI stained total DNA. (i) Representative confocal images of nuclei in E10.5 control (top right) and 78dko TGCs

Article Snippet: After antigen retrieval using Target Retrieval Solution (DAKO S1699), 5 μm deparaffinized sections of placenta or liver tissues were incubated with primary antibodies at 4 ◦C overnight against E2F7 1:100 (Abcam ab56022), E2F8 1:50 (polyclonal against residues 576–595 of murine E2F8), BrdU 1:100 (DAKOMO-0744), Ki67 1:100 (BD Pharmingen 550609), phospho-histone 3 (Ser10) 1:100 (Millipore 06-570), cyclin A2 1:100 (Santa Cruz sc-596), cyclin B1 1:100 (Santa Cruz sc-752), E-cadherin 1:100 (Abcam ab53033), p57Kip2 1:100 (Santa Cruz sc-8298; refs 56,57) and placental lactogen 1:200 (PL-1, F. Talamantes).

Techniques: Expressing, Immunohistochemistry, Mutagenesis, Staining, Control, Immunostaining, Microscopy

Journal: Nature cell biology

Article Title: Canonical and atypical E2Fs regulate the mammalian endocycle.

doi: 10.1038/ncb2595

Figure Lengend Snippet: Figure 4 Canonical activator and atypical repressor E2Fs regulate key transcriptional networks coordinating endocycles. (a) Heat map of approximately 4,500 differentially expressed genes in weaning age (3 wk) livers. Class I: genes regulated by E2F7 and E2F8. Class II: genes regulated by either E2F7 or E2F8. Class III: genes synergistically regulated by E2F7/E2F8. n = 3–4 livers analysed per genetic group. Control, E2f7f / f;E2f8f / f; Alb-78dko, Alb–cre;E2f7f / f;E2f8f/ f; Alb-8ko, Alb–cre;E2f8f / f; Alb-7ko, Alb–cre;E2f7f / f. (b) Top, heat map of TGC gene expression in a custom NanoString mRNA code set. RNA was isolated from laser-capture-microdissected E10.5 TGCs in frozen

Article Snippet: After antigen retrieval using Target Retrieval Solution (DAKO S1699), 5 μm deparaffinized sections of placenta or liver tissues were incubated with primary antibodies at 4 ◦C overnight against E2F7 1:100 (Abcam ab56022), E2F8 1:50 (polyclonal against residues 576–595 of murine E2F8), BrdU 1:100 (DAKOMO-0744), Ki67 1:100 (BD Pharmingen 550609), phospho-histone 3 (Ser10) 1:100 (Millipore 06-570), cyclin A2 1:100 (Santa Cruz sc-596), cyclin B1 1:100 (Santa Cruz sc-752), E-cadherin 1:100 (Abcam ab53033), p57Kip2 1:100 (Santa Cruz sc-8298; refs 56,57) and placental lactogen 1:200 (PL-1, F. Talamantes).

Techniques: Control, Gene Expression, Isolation

Journal: Nature cell biology

Article Title: Canonical and atypical E2Fs regulate the mammalian endocycle.

doi: 10.1038/ncb2595

Figure Lengend Snippet: Figure 5 Atypical repressors E2F7/E2F8 directly bind gene targets involved in endocycle control. (a) Immunoblot of transfected HepG2 cells showing exogenous expression of Flag-tagged E2F7 and E2F8 proteins with tubulin as the control. The arrows indicate tagged protein. (b) Representative ChIP assay in transfected HepG2 cells with anti-Flag antibodies demonstrating enhanced occupancy of Flag-tagged E2F7 and E2F8 proteins on E2F-binding sites in the promoter of G1/S (E2f1 and Ccne2) and G2/M (Ccna2 and Chek1) genes. The tubulin (Tub) gene, a non-E2F target, demonstrates specific recruitment of Flag-tagged E2F7 and E2F8 to target promoters containing consensus E2F-binding sequences. n = 3 independent transfection–ChIP experiments performed. (c) Immunoblot of transfected Rcho-1 trophoblast stem (TS) cells showing exogenous expression of Flag-tagged E2F7 and E2F8 proteins with tubulin as the control. The arrows indicate tagged protein. (d) Representative ChIP assays in transfected Rcho-1 trophoblast stem cells of G1/S and

Article Snippet: After antigen retrieval using Target Retrieval Solution (DAKO S1699), 5 μm deparaffinized sections of placenta or liver tissues were incubated with primary antibodies at 4 ◦C overnight against E2F7 1:100 (Abcam ab56022), E2F8 1:50 (polyclonal against residues 576–595 of murine E2F8), BrdU 1:100 (DAKOMO-0744), Ki67 1:100 (BD Pharmingen 550609), phospho-histone 3 (Ser10) 1:100 (Millipore 06-570), cyclin A2 1:100 (Santa Cruz sc-596), cyclin B1 1:100 (Santa Cruz sc-752), E-cadherin 1:100 (Abcam ab53033), p57Kip2 1:100 (Santa Cruz sc-8298; refs 56,57) and placental lactogen 1:200 (PL-1, F. Talamantes).

Techniques: Control, Western Blot, Transfection, Expressing, Binding Assay

Journal: Nature cell biology

Article Title: Canonical and atypical E2Fs regulate the mammalian endocycle.

doi: 10.1038/ncb2595

Figure Lengend Snippet: Figure 7 Cyclin A ablation reinstates genome ploidy of E2f7/E2f8-deficient TGCs and hepatocytes. (a) Images of H&E-stained E10.5 TGCs. The combined loss of Ccna1 and Ccna2 (Plf-A12dko) led to TGCs similar in appearance to control TGCs. The further loss of Ccna1 and Ccna2 in E2f7/E2f8-deficient TGCs (Plf-A1278qko) led to TGCs with a normal (wild-type-like) appearance, in contrast to Plf-78dko TGCs that seemed small or were binucleated (yellow arrows). The lower left insets show higher magnifications of the areas outlined in the main panels. The lower right insets show higher magnifications of the areas outlined in the main panels. (b) Feulgen quantification of genome ploidy in E10.5 TGCs. The intensities (that is, estimated genome content) of 120–140 TGCs were quantified per placenta sample, with n placenta samples analysed per genetic group as indicated. A number of TGCs quadruply deficient for Ccna1, Ccna2, E2f7 and E2f8 reach ploidy levels of 128C and 256C. Control, E2f7f / f;E2f8f / f; Plf-A12, Plfcre/+;Ccna1−/−;Ccna2f / f; Plf-78dko, Plfcre/+;E2f7f / f;E2f8f / f;Plf-A1278qko, Plfcre/+;Ccna1−/−;Ccna2f / f; E2f7f / f;E2f8f / f. (c) Images of H&E-stained 3-month-old liver sections,

Article Snippet: After antigen retrieval using Target Retrieval Solution (DAKO S1699), 5 μm deparaffinized sections of placenta or liver tissues were incubated with primary antibodies at 4 ◦C overnight against E2F7 1:100 (Abcam ab56022), E2F8 1:50 (polyclonal against residues 576–595 of murine E2F8), BrdU 1:100 (DAKOMO-0744), Ki67 1:100 (BD Pharmingen 550609), phospho-histone 3 (Ser10) 1:100 (Millipore 06-570), cyclin A2 1:100 (Santa Cruz sc-596), cyclin B1 1:100 (Santa Cruz sc-752), E-cadherin 1:100 (Abcam ab53033), p57Kip2 1:100 (Santa Cruz sc-8298; refs 56,57) and placental lactogen 1:200 (PL-1, F. Talamantes).

Techniques: Staining, Control