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. 2022 Feb 4;17:15. doi: 10.1186/s13024-021-00513-9

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Characterization of granulin antibodies and analysis of granulin levels in tissue lysates. a HEK293T lysates containing GFP or GFP tagged mouse granulins were probed with antibodies against each individual granulin peptide as indicated. Diagram of PGRN structure was shown at the top. b Western blot analysis of different tissues lysates from 4 to 5 months old WT and Grn^−/− mice with antibodies against each individual granulin, granulin A (GRN-A), granulin B (GRN-B), granulin C (GRN-C), granulin E (GRN-E) and granulin F (GRN- F) as indicated. Full length PGRN and total PGRN-derived granulins (GRNs) were detected by commercial sheep anti-mouse PGRN antibodies (R&D). Mixed male and female mice were used for this analysis. c Quantification of experiment in (b). The ratio between PGRN and GAPDH; granulins and GAPDH and between granulins and full-length PGRN was quantified and normalized to that in the liver sample on the same gel (set as 1). Data presented as mean ± SEM. n = 3. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001, unpaired two-tailed Student’s t-test. d Quantification of the ratios between granulins B/C/E/F and granulin A. The band intensities of different granulins were measured and normalized to GAPDH. The ratios between granulins B/C/E/F and granulin A were calculated and normalized to the lung sample on the same gel (set as 1). Data presented as mean ± SEM. The ratio of GRN-C (p = 0.04), GRN-E (p = 0.0057), GRN-F (p = 0.0006), but not GRN-B (p = 0.4586) versus GRN-A is significantly lower in kidney than that in lung. *, p < 0.05, **, p < 0.01, ***, p < 0.001, unpaired two-tailed Student’s t-test. e Granulin B, C, and E are glycosylated. Spleen or liver lysates from WT mice were immunoprecipitated using anti-granulin B, C, or E antibodies and the immunoprecipitates were treated with PNGase F. f Quantification of two differentially glycosylated forms of granulin C for experiments in (b). The ratio between top or bottom bands of granulin C and GAPDH in liver and spleen lysates was quantified and normalized to their levels in the lung. Data presented as mean ± SEM. n = 4. Top bands (p = 0.0023), bottom bands (p = 0.04), *, p < 0.05, **, p < 0.01, unpaired two-tailed Student’s t-test