Journal: FASEB BioAdvances

Article Title: The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

doi: 10.1096/fba.2021-00059

Figure Lengend Snippet: Expression and phosphorylation of Stat3 and Stat5. (A–C) Stat3 is phosphorylated and accumulates in the nucleus at peak lactation (day 10) in Btn1a1 −/− mice. (A) pStat3 was detected in sections of paraffin‐embedded mammary gland from (a) Btn1a1 +/+ , (b) Btn1a1 +/− , and (c) Btn1a1 −/− (lactation day 10, L10) and (d) Btn1a1 +/+ (involution day 1, Inv1) mice by the ABC VectorStain procedure as described in Materials and Methods. Representative examples of each genotype (3–6 sections each from three mice/genotype). Bar 100 µm. (B) Expression of Stat3 and phosphorylated Stat3 monitored by immunoblot of THs (60 µg protein/lane; 10% polyacrylamide) from 5 Btn1a1 +/+ , 7 Btn1a1 −/− mice at lactation days 9‐11 (L9‐11) and three each, Btn1a1 ++ mice at involution days 1 and 2 (Inv1 and Inv 2, respectively). pStat3 was detected with a 1000‐fold dilution of rabbit anti‐peptide‐Y705 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. Total Stat3 was detected on the same stripped blot with a 1000‐fold dilution of rabbit anti‐ C‐terminal Stat3‐peptide antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. (C) Triplicate blots as in B were quantified and data normalized to the amount of pStat3 in the Inv1 samples (set to 100%). pStat3/ Stat3 ratios are given below the graph. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA). (D–F) Stat5 is phosphorylated and accumulates in the nucleus at peak lactation (day 10) in Btn1a1 +/+ , Btn1a1 +/− , and Btn1a1 −/− mice. (D) pStat5 was detected in sections of paraffin‐embedded mammary gland as in A–C. Representative examples of each genotype (3–5 sections each from three mice/genotype). Alveoli denuded of epithelial cells (alveolar “ghosts”) discussed in the text are marked by asterisks in the knockout sample. Bar 100 µm. (E) Expression of Stat5 and phosphorylated Stat5 monitored by immunoblot of THs as in B above. Phosphorylated Stat5 (pStat5) was detected with a 1000‐fold dilution of rabbit anti‐peptide‐Y694 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. Total Stat5 was detected on the same stripped blot with a 1000‐fold dilution of rabbit anti‐peptide Stat5 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. (F) Duplicate blots as in E were quantified and data normalized to the amount of pStat5 in the Btn1a1 ++ L10 samples (set to 100%). pStat5/ Stat5 ratios are given below the graph. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA)

Article Snippet: Sections (7 µm) were dewaxed with Histoclear (HS‐200, National Diagnostics), rehydrated through a descending series of ethanol/water mixtures, and then immersed in 10 mM citrate buffer, pH 6.0 to retrieve antigen by heating in a pressure cooker for 1 min. Endogenous peroxidase was quenched by incubating the sections in 0.6% H 2 O 2 / 100% methanol for 30 min and the sections then rehydrated in water for 5 min. After blocking with 10% normal goat serum for 1 h, sections were incubated with 1‐ to 100‐fold dilutions of primary antibodies to either pStat3, pStat5, or Ki67 overnight at 4°C, followed by 1‐ to 50‐fold dilutions of mouse biotinylated anti‐rabbit IgG and avidin‐HRP, according to the ABC VectorStain procedure (Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Western Blot, Knock-Out

Journal: FASEB BioAdvances

Article Title: The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

doi: 10.1096/fba.2021-00059

Figure Lengend Snippet: The number (%) of mitotic epithelial cells increases at peak lactation in Btn1a1 −/− mice. (A) Ki67 was detected in sections of paraffin‐embedded mammary gland from (a) Btn1a1 +/+ , (b) Btn1a1 +/− , and (c) Btn1a1 −/− (lactation day 10, L10) by the ABC VectorStain procedure, as described in Materials and Methods. The number of Ki67‐positive nuclei was significantly increased in luminal Btn1a1 −/− cells compared with cells in Btn1a1 +/+ and Btn1a1 +/− mice (arrowheads). Representative examples of each genotype (4–6 sections, each from five mice/genotype). Bar 100 µm. (B) Section of embryonic day‐18 mouse mammary gland used as a positive control for Ki67 staining (1 of 5 examples). (C) Comparison of the number (%) of Ki67‐positive luminal cells at day 10 of lactation in Btn1a1 +/+ , Btn1a1 +/− , and Btn1a1 −/− mice (respective number of cells counted/genotype, 2,979, 2,529, and 1,418). Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA)

Article Snippet: Sections (7 µm) were dewaxed with Histoclear (HS‐200, National Diagnostics), rehydrated through a descending series of ethanol/water mixtures, and then immersed in 10 mM citrate buffer, pH 6.0 to retrieve antigen by heating in a pressure cooker for 1 min. Endogenous peroxidase was quenched by incubating the sections in 0.6% H 2 O 2 / 100% methanol for 30 min and the sections then rehydrated in water for 5 min. After blocking with 10% normal goat serum for 1 h, sections were incubated with 1‐ to 100‐fold dilutions of primary antibodies to either pStat3, pStat5, or Ki67 overnight at 4°C, followed by 1‐ to 50‐fold dilutions of mouse biotinylated anti‐rabbit IgG and avidin‐HRP, according to the ABC VectorStain procedure (Vector Laboratories, Burlingame, CA).

Techniques: Positive Control, Staining

Journal: FASEB BioAdvances

Article Title: The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

doi: 10.1096/fba.2021-00059

Figure Lengend Snippet: Expression and phosphorylation of Stat3 and Stat5. (A–C) Stat3 is phosphorylated and accumulates in the nucleus at peak lactation (day 10) in Btn1a1 −/− mice. (A) pStat3 was detected in sections of paraffin‐embedded mammary gland from (a) Btn1a1 +/+ , (b) Btn1a1 +/− , and (c) Btn1a1 −/− (lactation day 10, L10) and (d) Btn1a1 +/+ (involution day 1, Inv1) mice by the ABC VectorStain procedure as described in Materials and Methods. Representative examples of each genotype (3–6 sections each from three mice/genotype). Bar 100 µm. (B) Expression of Stat3 and phosphorylated Stat3 monitored by immunoblot of THs (60 µg protein/lane; 10% polyacrylamide) from 5 Btn1a1 +/+ , 7 Btn1a1 −/− mice at lactation days 9‐11 (L9‐11) and three each, Btn1a1 ++ mice at involution days 1 and 2 (Inv1 and Inv 2, respectively). pStat3 was detected with a 1000‐fold dilution of rabbit anti‐peptide‐Y705 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. Total Stat3 was detected on the same stripped blot with a 1000‐fold dilution of rabbit anti‐ C‐terminal Stat3‐peptide antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. (C) Triplicate blots as in B were quantified and data normalized to the amount of pStat3 in the Inv1 samples (set to 100%). pStat3/ Stat3 ratios are given below the graph. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA). (D–F) Stat5 is phosphorylated and accumulates in the nucleus at peak lactation (day 10) in Btn1a1 +/+ , Btn1a1 +/− , and Btn1a1 −/− mice. (D) pStat5 was detected in sections of paraffin‐embedded mammary gland as in A–C. Representative examples of each genotype (3–5 sections each from three mice/genotype). Alveoli denuded of epithelial cells (alveolar “ghosts”) discussed in the text are marked by asterisks in the knockout sample. Bar 100 µm. (E) Expression of Stat5 and phosphorylated Stat5 monitored by immunoblot of THs as in B above. Phosphorylated Stat5 (pStat5) was detected with a 1000‐fold dilution of rabbit anti‐peptide‐Y694 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. Total Stat5 was detected on the same stripped blot with a 1000‐fold dilution of rabbit anti‐peptide Stat5 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. (F) Duplicate blots as in E were quantified and data normalized to the amount of pStat5 in the Btn1a1 ++ L10 samples (set to 100%). pStat5/ Stat5 ratios are given below the graph. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA)

Article Snippet: Sections (7 µm) were dewaxed with Histoclear (HS‐200, National Diagnostics), rehydrated through a descending series of ethanol/water mixtures, and then immersed in 10 mM citrate buffer, pH 6.0 to retrieve antigen by heating in a pressure cooker for 1 min. Endogenous peroxidase was quenched by incubating the sections in 0.6% H 2 O 2 / 100% methanol for 30 min and the sections then rehydrated in water for 5 min. After blocking with 10% normal goat serum for 1 h, sections were incubated with 1‐ to 100‐fold dilutions of primary antibodies to either pStat3, pStat5, or Ki67 overnight at 4°C, followed by 1‐ to 50‐fold dilutions of mouse biotinylated anti‐rabbit IgG and avidin‐HRP, according to the ABC VectorStain procedure (Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Western Blot, Knock-Out

Journal: FASEB BioAdvances

Article Title: The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

doi: 10.1096/fba.2021-00059

Figure Lengend Snippet: Cathepsin B distribution in Btn1a1 +/+ , Btn1a1 +/− , and Btn1a1 −/− mammary cells at peak lactation (day 10). (A) Sections of paraffin‐embedded mammary gland from (a) Btn1a1 +/+ , (b) Btn1a1 +/− , and (c) two examples, Btn1a1 −/− (lactation day 10, L10) and (d) Btn1a1 +/+ (involution day 1, Inv1) mice were double‐labeled with antibodies to cathepsin B (Cts B) (Thermo) and lamp2 as described in Materials and Methods. Representative examples of each genotype (2–3 sections each from 3 mice/genotype) are shown for cathepsin B, lamp2 and the merged images. Note cathepsin B‐positive structures in Btn1a1 −/− and Btn1a1 +/+ Inv1 mice (arrowheads). (B) Expression of cathepsin B monitored by immunoblot of THs (75 µg protein/lane; 10% polyacrylamide) from Btn1a1 +/+ , Btn1a1 +/− , Btn1a1 −/− mice at lactation days 9–11 (L9‐11) (three mice/genotype) and three each, Btn1a1 ++ mice at involution days 1 and 2 (Inv1 and Inv 2, respectively). Cathepsin B was detected with a 1000‐fold dilution of monoclonal rabbit anti‐(human cathepsin B) (Abcam), followed by a 3,000‐fold dilution of goat anti‐(rabbit IgG)‐HRP. Pro‐cathepsin B and active forms are indicated to the right of the figure. Total cathepsin B was quantified on four blots to generate duplicate data for seven Btn1a1 +/+ , five Btn1a1 +/− , and six Btn1a1 −/− mice on lactation days 9–11 (L9‐11) and quadruplicate data for three mice, each on days 1 and 2 of involution (Inv1, and Inv2, respectively). Amounts of cathepsin B were normalized to the most positive Inv 2 Btn1a1 +/+ mouse (set to 100%) in the graph to the right of the blot. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA). Brightness in all micrographs was increased by 20% because of low fluorescence signals in the wild type and heterozygote samples. Asterisks, alveolar luminae, Bars 20 µm

Article Snippet: Sections (7 µm) were dewaxed with Histoclear (HS‐200, National Diagnostics), rehydrated through a descending series of ethanol/water mixtures, and then immersed in 10 mM citrate buffer, pH 6.0 to retrieve antigen by heating in a pressure cooker for 1 min. Endogenous peroxidase was quenched by incubating the sections in 0.6% H 2 O 2 / 100% methanol for 30 min and the sections then rehydrated in water for 5 min. After blocking with 10% normal goat serum for 1 h, sections were incubated with 1‐ to 100‐fold dilutions of primary antibodies to either pStat3, pStat5, or Ki67 overnight at 4°C, followed by 1‐ to 50‐fold dilutions of mouse biotinylated anti‐rabbit IgG and avidin‐HRP, according to the ABC VectorStain procedure (Vector Laboratories, Burlingame, CA).

Techniques: Labeling, Expressing, Western Blot, Fluorescence

Journal: FASEB BioAdvances

Article Title: The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

doi: 10.1096/fba.2021-00059

Figure Lengend Snippet: Determination of the amounts of Btn1a1 and Xdh in the lactating mammary glands of Btn1a1 +/+ , Btn1a1 +/− , and Btn1a1 −/− mice by immunoblot. A‐C. THs of mammary tissue, at days 9–11 of lactation, from seven Btn1a1 +/+ , five Btn1a1 +/− , and six Btn1a1 −/− mice and M r ‐marker proteins were separated by SDS–polyacrylamide gel electrophoresis (75 µg protein/lane; 10% polyacrylamide) and electroblotted to nitrocellulose. (A) Ponceau‐S stained blot. (B) Btn1a1 detected with a 5000‐fold dilution of rabbit anti‐peptide antibody to mouse Btn1a1 (Table ), followed by a 3000‐fold dilution of goat anti‐rabbit IgG‐HRP conjugate. Exposure was under nonlinear conditions to reveal the minor amounts of immunoreactive peptide bound to the 150 kDa‐band of Xdh in the Btn1a1 −/− samples (arrowhead). (C) Xdh detected in the same stripped blot with a 5000‐fold dilution of rabbit anti‐peptide antibody to mouse Xdh (Table ), followed by a 3,000‐fold dilution of goat anti‐rabbit IgG‐HRP conjugate. (D–F) Quantitation of the amounts of Btn1a1 and Xdh in triplicate blots under non‐saturating conditions. (D, E) Relative amounts of Btn1a1, viz , D the major 66 kDa‐band of Btn1a1 in B, and E, the Btn1a1 peptide bound to Xdh (arrowhead in B), normalized to the 66 kDa‐band in the Btn1a1 +/+ samples, set as 100%. Green columns show the same data corrected for the increases of Xdh noted in F below. See text for further discussion. (F) Amounts of Xdh in the same blots used for Btn1a1 analysis, stripped of residual antibodies, and normalized to Btn1a1 +/+ (set as 100%). There was a 3.6‐fold increase of Xdh in Btn1a1 +/− samples, and a sixfold increase in Btn1a1 −/− samples compared with the amount in Btn1a1 +/+ mice. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA)

Article Snippet: Sections (7 µm) were dewaxed with Histoclear (HS‐200, National Diagnostics), rehydrated through a descending series of ethanol/water mixtures, and then immersed in 10 mM citrate buffer, pH 6.0 to retrieve antigen by heating in a pressure cooker for 1 min. Endogenous peroxidase was quenched by incubating the sections in 0.6% H 2 O 2 / 100% methanol for 30 min and the sections then rehydrated in water for 5 min. After blocking with 10% normal goat serum for 1 h, sections were incubated with 1‐ to 100‐fold dilutions of primary antibodies to either pStat3, pStat5, or Ki67 overnight at 4°C, followed by 1‐ to 50‐fold dilutions of mouse biotinylated anti‐rabbit IgG and avidin‐HRP, according to the ABC VectorStain procedure (Vector Laboratories, Burlingame, CA).

Techniques: Western Blot, Marker, Polyacrylamide Gel Electrophoresis, Staining, Quantitation Assay