Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 (pSTAT3), AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Phospho-proteomics, Protein-Protein interactions, Ligation

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA mapping of signaling interactions in a lymph-node Hodgkin lymphoma, mixed cellularity (right neck); Hodgkin lymphoma, lymphocyte-depleted (neck); Hodgkin lymphoma, lymphocyte-predominant (left neck); Hodgkin lymphoma, mixed cellularity (left neck); and thymoma type B3 (mediastinum). Top row (visualization cycle 1) displays MEK1–ERK2 (FITC), EGFR–GRB2 (Cy5) and GRB2–MEK1 (Cy3N) together with DAPI. Middle row (cycle 2) shows STAT3–STAT5a (FITC), JAK1–JAK3 (Cy5) and JAK1–PI3Kp85 (Cy3N). Bottom row (cycle 3) presents JAK2– STAT5a (FITC), JAK2–JAK3 (Cy5) and JAK1–STAT3 (Cy3N). All nine pairs of antibody-oligonucleotide conjugates were applied and then amplified in a single incubation. The RCA products were revealed using detection oligonucleotides conjugated with three fluorophores in three visualization cycles. A standard three-channel fluorescence microscope was used with identical settings for all three fluorophores. Scale bars, 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Amplification, Incubation, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: Analysis of primary blood cells from two patients diagnosed with CML, targeting molecular pathways known to be up-regulated in CML. The experiment used a slightly different oligonucleotide design compared to other experiments reported herein, but with similar performance ( .) A, E) Quantitative analysis of numbers of signals per cell revealed striking differences among individual blood cells. Top and bottom rows represent data for two different CML patients. Visualization cycle 1: phosphoPI3K-AKT1 in Cy3 (red), phosphoAKT1-AKT3 in Cy5 (green), AKT(pan)-AKT2 in FITC (yellow). Cycle 2: JAK2-JAK3 in Cy3 (blue), phosphorylated GRB2 in Cy5 (orange), MEK1-ERK in FITC (purple). Cycle 3: STAT3-phosphoSTAT3 in Cy5 (cyan), STAT3-phosphoSTAT3 in FITC (magenta) and STAT3-STAT5 in Cy3 (lime). The same color coding was used throughout all panels in the figure. B, F) Scatterplots of pairs of detection reactions, serving to visualize correlations across detection pairs (colors as in A, B)). C, G) Visualization of a zoomed-in view of detection reactions per cell. Outline of nuclei are shown in red. D, H) Cell to cell heterogeneity is visualized by overlaying individual detection reactions on the cells - here showing zoomed in region with D) phosphorylated GRB2 (in orange) and (h) STAT3-phosphoSTAT3 (in magenta). Cell nuclei are outlined in red. The raw image data from the full sample, together with detections are available for interactive viewing at https://ulflandegren2025.serve.scilifelab.se .

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques:

Journal: Endocrinology

Article Title: Role of the Beta and Gamma Isoforms of the Adapter Protein SH2B1 in Regulating Energy Balance.

doi: 10.1210/endocr/bqad032

Figure Lengend Snippet: Figure 1. Generation and validation of neuron-specific SH2B1βγ NKO mouse model. (A) Schematic of SH2B1 isoforms. The isoform-specific C-terminal tails are denoted by the rectangles after residue 631. Numbers indicate amino acid residues in mouse and human sequences. P, proline-rich domain; DD, dimerization domain; NLS, nuclear localization sequence; NES, nuclear export sequence; PH, pleckstrin homology domain; SH2, Src homology domain. (B) Schematic showing region of Sh2b1 gene to be deleted to prevent Continued

Article Snippet: Equal amounts of protein in tissue lysates were subjected to immunoblotting overnight with mouse monoclonal antibody to SH2B1 (1:1000) (cat. no. sc-136065; Santa Cruz; RRID:AB_2301871) in 10 mM Tris, 150 mM NaCl, pH 7.4 (tris-buffered saline [TBS]) containing 0.1% Tween 20 and 1% fish gel (cat. no. G7041; Sigma), followed by IRDye 680LT goat antimouse antibody (1:15 000) (cat. no. 926-68020; Li-Cor; RRID: AB_10706161) in TBS containing 0.005% sodium dodecyl sulfate for 1 hour at room temperature.

Techniques: Biomarker Discovery, Residue, Sequencing

Journal: Endocrinology

Article Title: Role of the Beta and Gamma Isoforms of the Adapter Protein SH2B1 in Regulating Energy Balance.

doi: 10.1210/endocr/bqad032

Figure Lengend Snippet: Figure 4. Generation and validation of whole-body SH2B1βγ KO mouse model. (A) Schematic showing the Sh2b1 gene with exon 9 deleted due to nonhomologous enjoining (NHEJ) occurring as a byproduct of CRISPR/Cas9 editing designed to insert loxP sites flanking Exon 9. The arrow denotes the location of the NHEJ. (B) Genomic DNA was purified from mouse tails and analyzed by PCR using primers indicated in Panels 1B, 4A, and Table 3. (C) Proteins in lysates of whole brain, pancreas, or liver from WT or SH2B1 βγ KO (18 weeks old) littermates or SH2B1 KO mice were immunoblotted with αSH2B1 and reprobed with αβ-actin as indicated. The migration of molecular weight standards is shown on the right. The expected migrations of the different isoforms of SH2B1 and β-actin are indicated on the left. IB, immunoblot; n.s., nonspecific band.

Article Snippet: Equal amounts of protein in tissue lysates were subjected to immunoblotting overnight with mouse monoclonal antibody to SH2B1 (1:1000) (cat. no. sc-136065; Santa Cruz; RRID:AB_2301871) in 10 mM Tris, 150 mM NaCl, pH 7.4 (tris-buffered saline [TBS]) containing 0.1% Tween 20 and 1% fish gel (cat. no. G7041; Sigma), followed by IRDye 680LT goat antimouse antibody (1:15 000) (cat. no. 926-68020; Li-Cor; RRID: AB_10706161) in TBS containing 0.005% sodium dodecyl sulfate for 1 hour at room temperature.

Techniques: Biomarker Discovery, CRISPR, Purification, Migration, Molecular Weight, Western Blot

Journal: Journal of cell science

Article Title: The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

doi: 10.1242/jcs.259179

Figure Lengend Snippet: Fig. 2. SH2B1 isoforms increase neurite complexity and/or length. (A–C) Parameters measured using Simple Neurite Tracer on images of Sh2b1 KO hippocampal neurons transiently expressing GFP (−) or GFP–SH2B1 isoforms. Neurons were transfected (DIV4) and imaged (DIV5) using fluorescence microscopy. The number (n) of neurons from three distinct experiments was: GFP, n=44; GFP–SH2B1α, n=45; GFP–SH2B1β, n=48; GFP–SH2B1γ, n=46; GFP–SH2B1δ, n=44. (D–F) Parameters obtained from Sholl analysis of images used in A–C. D inset, subset of data in D. Data are means±s.e.m. Statistics: A–C, E,F, one-tailed unpaired t-test, WT versus KO neurons expressing GFP (#P<0.05); one-way ANOVA with Dunnett’s multiple comparisons test, KO neurons expressing GFP versus each GFP–SH2B1 isoform (*P<0.05); D, inset, two-way repeated measures ANOVA with Dunnett’s multiple comparisons test, KO neurons expressing GFP versus each GFP–SH2B1 isoform, thick lines indicate significance (*P<0.05).

Article Snippet: Mousemonoclonal antibody to SH2B1 (sc-136065; 1:1000) was from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Transfection, Fluorescence, Microscopy, One-tailed Test

Journal: Journal of cell science

Article Title: The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

doi: 10.1242/jcs.259179

Figure Lengend Snippet: Fig. 3. The ability of SH2B1δ to increase neurite complexity is altered by some human obesity-associated SH2B1 variants. (A) Schematic of SH2B1δ showing human obesity-associated variants. (B–D) Parameters measured using Simple Neurite Tracer on images of Sh2b1 KO hippocampal neurons transiently expressing GFP (−) or the indicated GFP–SH2B1δ construct. Neurons were transfected (DIV4) and imaged (DIV5) using fluorescence microscopy. The number (n) of neurons from three distinct experiments was: GFP, n=34; GFP–SH2B1δ(WT), n=45; GFP–SH2B1δ(P90H), n=38; GFP–SH2B1δ(P322S), n=39; GFP–SH2B1δ(A484T), n=38; GFP–SH2B1δ(T546A), n=43; GFP–SH2B1δ(R680C), n=39. (E) Sholl analysis of images used in B-D. Inset: subset of data in E. Data are means±s.e.m. Statistics: B–D, one-tailed unpaired t-test, GFP versus GFP–SH2B1δ(WT) (#P<0.05); one-way ANOVA with Dunnett’s multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ variant (no significant differences detected); E, inset, two-way repeated measures ANOVAwith Dunnett’s multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ variant, thick lines indicate significance (*P<0.05).

Article Snippet: Mousemonoclonal antibody to SH2B1 (sc-136065; 1:1000) was from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Construct, Transfection, Fluorescence, Microscopy, One-tailed Test, Variant Assay

Journal: Journal of cell science

Article Title: The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

doi: 10.1242/jcs.259179

Figure Lengend Snippet: Fig. 4. SH2B1δ localizes to nucleoli and the plasma membrane. (A) PC12 cells transiently co-expressing GFP–SH2B1 isoforms and mCherry–nucleolin were stained with Alexa Fluor 488–conjugated wheat germ agglutinin (WGA) and imaged using live-cell confocal microscopy. Left five panels: low photomultiplier gain. Right two panels: high photomultiplier gain. Images representative of ≥20 cells/isoform. DIC, differential interference contrast. (B) Sh2b1 KO hippocampal neurons were transiently transfected with the indicated GFP–SH2B1 isoforms (DIV5) and imaged (DIV6) using fluorescence (top panels) or confocal microscopy of fixed neurons stained with antibody to GFP (αGFP) (middle panels) or live-cell confocal microscopy (bottom panels). Images inverted in top and middle panels. Arrowheads (middle panels) denote microstructures protruding from dendritic shafts. Scale bars: 10 µm.

Article Snippet: Mousemonoclonal antibody to SH2B1 (sc-136065; 1:1000) was from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Clinical Proteomics, Membrane, Expressing, Staining, Confocal Microscopy, Transfection, Fluorescence

Journal: Journal of cell science

Article Title: The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

doi: 10.1242/jcs.259179

Figure Lengend Snippet: Fig. 5. SH2B1δ promotes NGF-stimulated signaling activities. (A) Serum-starved PC12 cells stably expressing GFP, GFP–SH2B1β or GFP–SH2B1δ were stimulated with 25 ng/ml NGF as indicated. Proteins in cell lysates were immunoblotted (IB) with the indicated antibodies. Migration of molecular mass standards is on the left. (B–D) Quantification of immunoblots in A plus two additional sets of immunoblots, each performed using a distinct biological sample. Relative signals of phosphorylated proteins were normalized first to the signal for the total amount of that protein and then to the signal for GFP-expressing cells stimulated with NGF for 10 min. Data are means±s.e.m. Statistics: B–D, one-way ANOVA at 10 and 60 min with Tukey’s multiple comparisons test to compare GFP versus GFP– SH2B1β (#P<0.05) and GFP versus GFP–SH2B1δ (*P<0.05).

Article Snippet: Mousemonoclonal antibody to SH2B1 (sc-136065; 1:1000) was from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Stable Transfection, Expressing, Migration, Western Blot

Journal: Journal of cell science

Article Title: The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

doi: 10.1242/jcs.259179

Figure Lengend Snippet: Fig. 6. Bipartite NLS in C-terminal tail is required for SH2B1δ to localize in nucleoli. (A) Schematic of SH2B1δ with mutations and truncation. Magenta font and/or underline indicates mutation or removal of amino acids. mNLS, mutated NLS. (B) PC12 cells transiently co-expressing the indicated GFP–SH2B1δ mutant and mCherry–nucleolin were stained with Alexa Fluor 467-conjugated WGA and imaged using live-cell confocal microscopy. Left five panels: low photomultiplier gain. Right two panels: high photomultiplier gain. DIC, differential interference contrast. Scale bars: 10 µm. (C) Subcellular localization of the indicated SH2B1δ mutants. Bright, dim and absent refer to the signal intensity in indicated cellular compartment [bright, seen with low gain; dim, seen only with high gain; absent, not seen even with high gain]. The number (n) of cells from two or three distinct experiments were: SH2B1δ(WT), n=28; δ(mNLS2), n=24; δ(mNLS3), n=19; δ(mNLS2+3), n=25; δ(W712X), n=20; δ(mNLS1), n=25. PM, plasma membrane.

Article Snippet: Mousemonoclonal antibody to SH2B1 (sc-136065; 1:1000) was from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Mutagenesis, Expressing, Staining, Confocal Microscopy, Clinical Proteomics, Membrane

Journal: Journal of cell science

Article Title: The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

doi: 10.1242/jcs.259179

Figure Lengend Snippet: Fig. 7. SH2B1δ must localize to nucleoli and the plasma membrane to maximally increase neurite complexity. (A) Schematic of SH2B1δ showing mutations. (B) Sh2b1 KO hippocampal neurons were transiently transfected (DIV5) with cDNA encoding the indicated GFP–SH2B1δ constructs and imaged (DIV6) using live-cell confocal microscopy. Left three panels: low photomultiplier gain. Right panel: high photomultiplier gain. Scale bars: 10 µm. (C–E) Parameters measured using Simple Neurite Tracer on images of KO neurons transiently expressing the indicated GFP–SH2B1δ construct. Neurons were transfected (DIV4) and imaged (DIV5) using fluorescence microscopy. The number (n) of neurons from three distinct experiments was: GFP, n=49; GFP–SH2B1δ(WT), n=58; GFP–SH2B1δ(mNLS2+3), n=41; GFP–SH2B1δ(W712X), n=28; GFP–SH2B1δ(mNLS1), n=27; GFP–SH2B1δ(R555E), n=34. (F) Sholl analysis of neuron images used in C–E. Inset: subset of data in F. Data are means±s.e.m. Statistics: C–E, one-tailed unpaired t-test, GFP versus GFP– SH2B1δ(WT) (#P<0.05); one-way ANOVA with Dunnett’s multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ mutant (*P<0.05); F, inset, two-way repeated measures ANOVA with Dunnett’s multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ mutant, thick lines indicate significance (*P<0.05).

Article Snippet: Mousemonoclonal antibody to SH2B1 (sc-136065; 1:1000) was from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Clinical Proteomics, Membrane, Transfection, Construct, Confocal Microscopy, Expressing, Fluorescence, Microscopy, One-tailed Test, Mutagenesis

Journal: Journal of cell science

Article Title: The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

doi: 10.1242/jcs.259179

Figure Lengend Snippet: Fig. 8. SH2B1δ enhances neurotrophin-induced expression of neuronal immediate early genes; enhancement is partially dependent on nucleolar SH2B1δ. (A–D) PC12 cells stably expressing GFP (−), GFP–SH2B1β or GFP–SH2B1δ were treated with or without NGF (100 ng/ml, 6 h). Relative mRNA levels of (A) Plaur, (B) Mmp3, (C) Mmp10 and (D) FosL1 were determined using qPCR. Data were normalized to NGF-treated GFP–SH2B1β values. n=3 biological replicates. (E,F) WT or KO hippocampal neurons (DIV5) were treated with or without BDNF (50 ng/ml, 1 h). Relative mRNA levels of (E) Egr1 and (F) Arc were determined using qPCR. n=3 biological replicates. (G–I) WT or KO neurons (DIV8) were infected with lentivirus encoding GFP, GFP–SH2B1β or GFP–SH2B1δ and treated with or without BDNF (50 ng/ml, 2 h) (DIV14). Relative mRNA levels of (G) FosL1, (H) Egr1 and (I) Arc were determined using qPCR. n=3−5 biological replicates; one set of biological replicates of GFP–SH2B1β (with or without BDNF) was averaged from duplicate samples. Data are means±s.e.m. Statistics: A–D, one-way ANOVA (randomized block experiment) on NGF-treated samples with Holm–Sidak’s multiple comparisons test (*P<0.05); one-way ANOVA (randomized block experiment) on untreated samples (no significant differences detected); E,F, one-tailed paired t-test on BDNF-treated samples (*P<0.05); one-tailed paired t-test on untreated samples (no significant differences detected); G–I, one-way ANOVA (randomized block experiment with mixed effects analysis) with Holm–Sidak’s multiple comparisons test on BDNF-treated samples, GFP versus GFP–SH2B1β versus GFP–SH2B1δ (*P<0.05); one-tailed paired t-test on BDNF-treated samples, GFP–SH2B1δ versus GFP–SH2B1δ(mNLS2+3) (#P<0.05); one-way ANOVA (randomized block experiment with mixed effects analysis) with Holm–Sidak’s multiple comparisons test on untreated samples (no significant differences detected).

Article Snippet: Mousemonoclonal antibody to SH2B1 (sc-136065; 1:1000) was from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Stable Transfection, Infection, Blocking Assay, One-tailed Test

Journal: Redox Biology

Article Title: Gestational zinc deficiency impairs brain astrogliogenesis in rats through multistep alterations of the JAK/STAT3 signaling pathway

doi: 10.1016/j.redox.2021.102017

Figure Lengend Snippet: Gestational MZD affects different steps in the STAT3 pathway in the offspring brain cortex at different developmental stages. Dams were fed ad libitum either a control (C) or a marginal zinc diet (MZD) from gestation day 0 until E14, E19 and P2, at which time dams, and subsequently (after P20) the offspring were fed a control diet until P56. A) Experimental design. B-M) Brain cortex homogenates were prepared as described in the Materials and methods section. Western blots for B-D ) phosphorylated STAT3 at tyrosine-705 (p Y 705 -STAT3), total STAT3, and GAPDH; E-G ) phosphorylated JAK2 at tyrosine-1007/1008 (p Y 1007/1008 -JAK2), total JAK2, and GAPDH; H-J ) PTP1B, SHP2 and GAPDH; and K-M ) CT-1, LIF and GAPDH. GAPDH was used as loading control. After quantifications of bands, values were calculated as the ratios C ) p Y 705 -STAT3/STAT3, D ) STAT3/GAPDH, F ) p Y 1007/1008 -JAK2/JAK2, G ) JAK2/GAPDH, I ) PTP1B/GAPDH, J ) SHP2/GAPDH, L ) CT-1//GAPDH and M ) LIF/GAPDH. For all proteins values were normalized to those of the E14 control group. Results are shown as means ± S.E.M and are the average of 6 litters per group per developmental stage. *, p ≤ 0.05; **, p ≤ 0.01 are significantly different compared to the respective control at each developmental stage (Student's t -test).

Article Snippet: Primary antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), leukemia inhibitory factor (LIF), SH2 containing protein tyrosine phosphatase-2 (SHP2), and S100 calcium-binding protein β (S100β), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), β-actin, and Protein A/G PLUS-Agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Western Blot

Journal: Redox Biology

Article Title: Gestational zinc deficiency impairs brain astrogliogenesis in rats through multistep alterations of the JAK/STAT3 signaling pathway

doi: 10.1016/j.redox.2021.102017

Figure Lengend Snippet: Gestational MZD alters the interactions of STAT3 with the tyrosine phosphatase PTP1B and cytoskeleton proteins in the E19 brain CT. A-C ) STAT3 was immunoprecipitated from E19 CT tissue lysates as described in the Materials and methods section. A) Western blots for PTP1B, SHP2, α-tubulin, β-actin and STAT3 in the STAT3 immunoprecipitates (IP). B ) Western blot for phosphotyrosine-705 STAT3 (p Y 705 -STAT3) and STAT3 I in the STAT3 immunoprecipitates (IP). C ) After quantifications of bands, values were calculated as the ratios p Y 705 -STAT3/STAT3, SHP2/STAT3, PTP1B/STAT3, α-tubulin/STAT3, and β-actin/STAT3, and normalized to control levels. D,E) Brain CT was subjected to non-reducing homogenization and SDS-PAGE. D ) Upper panel: Western blots for α-tubulin in control (C1,C2) and MZD (MZ1, MZ2) samples treated without (−) or with (+) addition of 1% (v/v) β-mercaptoethanol. St: molecular weight standard. HSC70 was assessed as loading control. E) After quantification, the ratio of intensity between bands of molecular weight higher than 100 kDa/50 kDa was calculated. Results are shown as means ± S.E.M. of six litters per group. *, p ≤ 0.05; **, p ≤ 0.01 are significantly different compared to controls (Student's t -test).

Article Snippet: Primary antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), leukemia inhibitory factor (LIF), SH2 containing protein tyrosine phosphatase-2 (SHP2), and S100 calcium-binding protein β (S100β), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), β-actin, and Protein A/G PLUS-Agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Western Blot, Control, Homogenization, SDS Page, Molecular Weight